CNX antigen-binding molecules

Abstract

Disclosed herein are antigen-binding molecules capable of binding to calnexin (CNX). Chimeric antigen receptors, antibody-drug conjugates, and compositions comprising these antigen-binding molecules, as well as nucleic acids, vectors, and cells, are also disclosed. Uses and methods involving antigen-binding molecules capable of binding to calnexin (CNX) are also disclosed.

Description

[Technical Field]

[0001] This application claims priority from U.S. Ser. No. 63 / 359,499, filed July 8, 2022, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to the field of molecular biology, and more particularly to antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis. [Background technology]

[0003] CNX is an endoplasmic reticulum (ER)-resident lectin chaperone protein that binds to N-glycoproteins carrying monoglycosylated glycans and recruits various other chaperones that mediate protein disulfide formation, proline isomerization, and protein folding.

[0004] Recent studies have implicated CNX and CNX-containing complexes (e.g., CNX:ERp57) in the pathogenesis of diseases and conditions, including cancer, particularly through their ECM-degrading activity (see Ros et al. Nat. Cell Biol. (2020) 22(11):1371-1381).

[0005] Ros et al., Nat. Cell Biol. (2020) 22(11):1371-1381, disclose anti-CNX antibodies, ab10286 and ab22595. Abcam's ab10286 and ab22595 are rabbit polyclonal antibody preparations against human CNX, respectively. There remains a need for the development of antibodies against CNX suitable for use in medical treatment and prophylaxis methods. Summary of the Invention [Means for solving the problem]

[0006] In a first aspect, the present disclosure provides an antigen-binding molecule, optionally isolated, that binds to CNX. In some embodiments, the antigen binding molecule inhibits the degradation of the extracellular matrix (ECM).

[0007] In some aspects, the antigen-binding molecule comprises: (a) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 166; HC-CDR2 having the amino acid sequence of SEQ ID NO: 167; HC-CDR3 having the amino acid sequence of SEQ ID NO: 168 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 179; LC-CDR2 having the amino acid sequence of SEQ ID NO: 180; LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 a light chain variable (VL) region incorporating: (b) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33; HC-CDR2 having the amino acid sequence of SEQ ID NO: 34; HC-CDR3 having the amino acid sequence of SEQ ID NO: 35 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 43 a light chain variable (VL) region incorporating: (c) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 4 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 10; LC-CDR2 having the amino acid sequence of SEQ ID NO: 11; LC-CDR3 having the amino acid sequence of SEQ ID NO: 12 a light chain variable (VL) region incorporating: (d) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 18; HC-CDR2 having the amino acid sequence of SEQ ID NO: 19; HC-CDR3 having the amino acid sequence of SEQ ID NO: 20 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 25; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 27 a light chain variable (VL) region incorporating: (e) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 49 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 53; LC-CDR2 having the amino acid sequence of SEQ ID NO: 54; LC-CDR3 having the amino acid sequence of SEQ ID NO: 55 a light chain variable (VL) region incorporating: (f) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 68; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 69 a light chain variable (VL) region incorporating: (g) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 74 a light chain variable (VL) region incorporating: (h) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 78; LC-CDR2 having the amino acid sequence of SEQ ID NO: 79; LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 a light chain variable (VL) region incorporating: (i) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 83 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 74 a light chain variable (VL) region incorporating: (j) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 86 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 89; LC-CDR2 having the amino acid sequence of SEQ ID NO: 11; LC-CDR3 having the amino acid sequence of SEQ ID NO: 90 a light chain variable (VL) region incorporating: (k) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 95; HC-CDR3 having the amino acid sequence of SEQ ID NO: 96 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 101; LC-CDR2 having the amino acid sequence of SEQ ID NO: 102; LC-CDR3 having the amino acid sequence of SEQ ID NO: 103 a light chain variable (VL) region incorporating: (l) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108; HC-CDR2 having the amino acid sequence of SEQ ID NO: 109; HC-CDR3 having the amino acid sequence of SEQ ID NO: 110 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 115; LC-CDR2 having the amino acid sequence of SEQ ID NO: 116; LC-CDR3 having the amino acid sequence of SEQ ID NO: 117 a light chain variable (VL) region incorporating: (m) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 122 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 125; LC-CDR2 having the amino acid sequence of SEQ ID NO: 126; LC-CDR3 having the amino acid sequence of SEQ ID NO: 127 a light chain variable (VL) region incorporating: (n) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 132; HC-CDR2 having the amino acid sequence of SEQ ID NO: 133; HC-CDR3 having the amino acid sequence of SEQ ID NO: 134 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 139; LC-CDR2 having the amino acid sequence of SEQ ID NO: 140; LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 a light chain variable (VL) region incorporating: (o) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 146; HC-CDR2 having the amino acid sequence of SEQ ID NO: 147; HC-CDR3 having the amino acid sequence of SEQ ID NO: 148 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 153 a light chain variable (VL) region incorporating: (p) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 156 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 158; LC-CDR2 having the amino acid sequence of SEQ ID NO: 159; LC-CDR3 having the amino acid sequence of SEQ ID NO: 160 a light chain variable (VL) region incorporating: (q) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 166; HC-CDR2 having the amino acid sequence of SEQ ID NO: 167; HC-CDR3 having the amino acid sequence of SEQ ID NO: 168 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 171; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 a light chain variable (VL) region incorporating: (r) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 186; HC-CDR3 having the amino acid sequence of SEQ ID NO: 187 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 194 a light chain variable (VL) region incorporating: (s) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 199; HC-CDR3 having the amino acid sequence of SEQ ID NO: 200 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 205; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 206 a light chain variable (VL) region incorporating: (t) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 211; HC-CDR3 having the amino acid sequence of SEQ ID NO: 212 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 216; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 217 a light chain variable (VL) region incorporating: (u) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 222; HC-CDR2 having the amino acid sequence of SEQ ID NO: 223; HC-CDR3 having the amino acid sequence of SEQ ID NO: 224 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 230 a light chain variable (VL) region incorporating: (v) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 199; HC-CDR3 having the amino acid sequence of SEQ ID NO: 200 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235; LC-CDR2 having the amino acid sequence of SEQ ID NO: 236; LC-CDR3 having the amino acid sequence of SEQ ID NO: 237 a light chain variable (VL) region incorporating: (w) (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 243; HC-CDR3 having the amino acid sequence of SEQ ID NO: 244 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 248; LC-CDR2 having the amino acid sequence of SEQ ID NO: 249; LC-CDR3 having the amino acid sequence of SEQ ID NO: 250 Light chain variable (VL) region incorporating Includes.

[0008] In some aspects, the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165, 32, 1, 17, 47, 60, 82, 85, 94, 107, 121, 131, 154, 155, 184, 198, 210, 221, or 242; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 178, 40, 9, 24, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 191, 204, 215, 228, 234, or 247; Includes.

[0009] In some aspects, the antigen-binding molecule comprises: (i) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 178; or (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 32; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 40; or (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9; or (iv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 24; or (v) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 47; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 52; or (vi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 67; or (vii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 72; or (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 77; or (ix) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of a SEQ ID NO:; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:; or (x) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 82; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 72; or (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 85; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 88; or (xii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 94; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 100; or (xiii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 107; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 114; or (xiv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 121; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 124; or (xv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 131; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 138; or (xvi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 145; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 152; or (xvii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 155; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 157; or (xviii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 170; or (xix) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 184; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 191; or (xx) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 204; or (xxi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 210; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 215; or (xxii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 221; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 228; or (xxiii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 234; or (xxiv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 242; and A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 247. Includes.

[0010] In some embodiments, the antigen-binding molecule (a) binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 363, and optionally binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 361 or 362, or (b) binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 371, and optionally binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 364, 365, 366, 367, 368, 369, 370, 372, or 373.

[0011] In some embodiments, the antigen-binding molecule binds to CRT. In some embodiments, the antigen-binding molecule binds to human CNX and mouse CNX. In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule, and the antigen-binding molecule further comprises an antigen-binding domain that binds to an antigen other than CNX. In some embodiments, the multispecific antigen-binding molecule is a bispecific T cell engager (BiTE).

[0012] The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present disclosure. The present disclosure also provides an antibody drug conjugate (ADC) comprising an antigen-binding molecule according to the present disclosure and a drug moiety.

[0013] The present disclosure also provides a nucleic acid or nucleic acids, optionally isolated, encoding an antigen-binding molecule or CAR according to the present disclosure. The present disclosure also provides an expression vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure.

[0014] The present disclosure also provides a cell comprising an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors according to the present disclosure. The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of the antigen binding molecule or CAR by the cell.

[0015] The present disclosure also provides a composition comprising an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, or a cell according to the present disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0016] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or vectors, a cell, or a composition according to the present disclosure for use in methods of medical treatment and prophylaxis.

[0017] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell, or a composition according to the present disclosure for use in a method of treating or preventing a disease / condition characterized by degradation of the extracellular matrix (ECM).

[0018] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell, or a composition according to the present disclosure for use in a method of treating or preventing cancer.

[0019] In some aspects, the cancer is selected from liver cancer, breast cancer, oral cancer, oral squamous cell carcinoma, sarcoma, lung cancer, prostate cancer, bladder cancer, renal cancer, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer, and thyroid cancer.

[0020] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell, or a composition according to the present disclosure for use in a method of treating or preventing cartilage degradation or a disease / condition characterized by cartilage degradation.

[0021] In some embodiments, the disease / condition characterized by cartilage degradation is selected from joint disorders, arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondrosis dissecans, cartilage damage, and polychondritis.

[0022] The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure to deplete or increase killing of cells expressing CNX. The present disclosure also provides an in vitro complex comprising an antigen-binding molecule according to the present disclosure that binds to CNX, optionally isolated.

[0023] The present disclosure also provides a method for detecting CNX in a sample, the method comprising contacting a sample containing or suspected of containing CNX with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex between the antigen-binding molecule and CNX.

[0024] The present disclosure also provides a method for selecting or stratifying a subject for treatment with an agent that targets CNX, the method comprising contacting a sample from the subject in vitro with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex between the antigen-binding molecule and CNX.

[0025] The present disclosure also provides the use of an antigen-binding molecule according to the invention as an in vitro or in vivo diagnostic or prognostic agent. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings. [Brief explanation of the drawings]

[0026] [Figure 1] Binding ELISA data of 15 Fab supernatant clones against the antigen protein of biotinylated recombinant human CANX protein (HuCANX_hFc) fused at the C-terminus to the Fc region of human IgG1. Fifteen Fab clones were tested in a binding ELISA assay to evaluate their antigen binding to biotinylated HUCANX with a human Fc tag and detected by goat anti-human Fab-HRP (horseradish peroxidase). [Figure 2] Polyclonal phage ELISA data after panning rounds 1, 2, and 3 against a purified fragment of human calnexin containing amino acids 1-481 fused to the human FC region (FC-CNX). Detection of bound polyclonal phage to wells coated with FC, FC-CNX, or BSA was performed with anti-M13-HRP antibody. [Figure 3] Monoclonal phage ELISA with the indicated clones tested on wells coated with FC-CNX, FC, or BSA. Detection was performed with anti-M13 HRP. [Figure 4] (A) Binding affinity ELISA of 15 IgG1s to the antigen protein of biotinylated HuCANX_hFc. Fifteen antibody clones were tested in a binding ELISA assay to evaluate binding to the target protein biotinylated HuCANX with a human Fc tag, and detected by streptavidin-HRP with an irrelevant IgG1 used as a negative control antibody. (B) Binding affinity ELISA of 15 IgG1s to the antigen protein of HuCANX_His. Fifteen antibody clones were tested in a binding ELISA assay to evaluate binding to the target protein HuCANX with a His tag, and detected by anti-His-HRP with an irrelevant IgG1 used as a negative control antibody. [Figure 5]Binding affinity ELISA data for 15 IgG1 antibody clones against the antigen protein of CNX-His. 15 antibody clones were tested in a binding ELISA assay to assess binding to CNX-His using anti-human Fc-HRP for detection. An irrelevant human IgG1 was used as a negative control antibody, and CNX ab10286 (Abcam) was used as a positive control. The results are split into two graphs with controls for comparison. The table on the right side of the figure shows the estimated EC50 for each antibody. [Figure 6] The binding affinity of 15 IgG1s to the antigen protein of MsCANX_His was measured by biolayer interferometry. The table shows the derived equilibrium dissociation constants (KD), association constants (Kon), and dissociation rate constants (Kdis) for the IgG test antibodies. The affinities of 2H5 and 5A3 could not be determined. [Figure 7] The binding affinity of five IgG1s to the antigen protein of human CNX_His was measured by biolayer interferometry. The table shows the derived equilibrium dissociation constants (KD), association constants (Kon), and dissociation rate constants (Kdis) for the IgG test antibodies. [Figure 8] Binding ELISA test of eight recombinant scFv clones to wells coated with HUCANX_hFc (100 ng and 10 ng) antigen protein and BSA control. Eight clones were tested in a binding ELISA assay with detection using anti-myc tag HRP. [Figure 9] Binding affinity of scFv clone 8 to the antigen protein human FC-CNX. Measurements were performed by biolayer interferometry. The table shows the derived equilibrium dissociation constant (KD), association constant (Kon), and dissociation rate constant (Kdis) for scFv clone 8. [Figure 10]Epitope binding of 15 antibody clones by biolayer interferometry (BLI) analysis. Light gray highlighted boxes represent antibodies with non-overlapping epitopes on the HUCANX_His protein. Dark gray highlighted boxes represent antibodies that share at least partially overlapping epitopes with other antibodies. [Figure 11] HDX of scFv clone 8 in the presence of FC-CNX. Each point represents a detected peptide sequence from N-terminus (left) to C-terminus (right) as determined by mass spectrometry. The deuteron difference between the deuterium-labeled and unlabeled samples at each time point is scored on the Y-axis. The figure includes SEQ ID NOs: 384-388. [Figure 12] HDX of FC-CNX in the presence of scFv clone 8. Each point represents the detected peptide sequence from N-terminus (left) to C-terminus (right) as determined by mass spectrometry. The deuteron difference between the deuterium-labeled and unlabeled samples at each time point is scored on the Y-axis. The figure includes SEQ ID NOs: 389-391. [Figure 13] HDX of the heavy chain of 1E1 in the presence of human CNX-His. Each point represents a detected peptide sequence from the N-terminus (left) to the C-terminus (right) as determined by mass spectrometry. The difference in deuterons between the deuterium-labeled and unlabeled samples at each time point is scored on the Y-axis. The figure includes SEQ ID NOs: 392-400. [Figure 14] HDX of the light chain of IgG1 1E1 in the presence of human CNX-His. Each point represents a detected peptide sequence from the N-terminus (left) to the C-terminus (right) as directed by mass spectrometry. The deuteron difference between the deuterium-labeled and unlabeled samples at each time point is scored on the Y-axis. The figure includes SEQ ID NOs: 401-404. [Figure 15] HDX of human CNX-His in the presence of IgG 1E1. Each point represents a detected peptide sequence from the N-terminus (left) to the C-terminus (right) as determined by mass spectrometry. The difference in deuterons between the deuterium-labeled and unlabeled samples at each time point is scored on the Y-axis. The figure includes SEQ ID NOs: 405-407. [Figure 16] Localization of IgG antibodies in an immunofluorescence setting. Autocontrast images of immunofluorescent staining of MDA-231 cells with the indicated human IgG1 antibodies and secondary detection with anti-human-linked Alexa fluorochromes and Hoechst nuclear stain. [Figure 17] Immunofluorescence staining of CNX with IgG. Representative images of polyclonal Huh7 CNX-mcherry cells (right image pair) stained with the indicated human IgG1 antibody and secondary detection with anti-human-linked Alexa fluorochrome (left image pair). Quantitative graphs are shown, subtracting the intensity produced by the antibody in Huh7 cells from the intensity produced in CNX-mcherry cells. [Figure 18] Immunofluorescence staining of CNX with scFv. Images of Huh7 CNX mcherry cells co-stained with the indicated scFv and secondary anti-myc-linked Alexa fluorochrome detection. The R2 correlation between the cytoplasmic intensity of Huh7 CNX-Mcherry cells and the scFv-detected anti-myc intensity was calculated and is shown on the right. [Figure 19] Immunoblot testing of antibodies. Detection of the presence of CNX / CRT on nitrocellulose membranes containing proteins transferred from SDS-PAGE loaded with cell extracts from untreated HeLa cells or transfected with siRNA against CNX or CRT. Each of the indicated IgG1s was incubated on the membrane, and bound antibody was detected with an anti-human HRP secondary antibody (top and middle). An anti-actin loading control is shown in the top row. Testing of IgG1 clone 8 for the detection of CNX / CRT on membranes containing proteins transferred from SDS-PAGE loaded with cell extracts from MDA-231 ERG2 and MDA-231 ERG2 CNX- / - is shown on the right. Immunoblots with the control CNX antibody ab238078 and CRT antibody ab92516 are presented. [Figure 20]Fluorescent gelatin layering assay with Huh7 cells seeded for 48 hours in the presence of 10 μg / ml IgG control or scFV clone 8. Representative fluorescent gelatin areas and the associated respective Hoechst images are shown, along with a graph showing the calculated normalized resolved area per cell in several fields of view. [Figure 21] Fluorescent gelatin layering assay with Huh7 cells seeded for 48 hours in the presence of 20 μg / ml IgG control, ab22595 CNX antibody, scFV clone 8, or the indicated IgG1. Representative fluorescent gelatin areas and accompanying Hoechst images are shown, along with a graph showing the calculated normalized resolved area per cell in several fields of view. Error bars indicate the standard error of the mean (SEM). [Figure 22] Collagen DQ degradation assay with 3T3 v-Src cells seeded for 48 hours in the presence of 20 μg / ml IgG control, ab22595 anti-CNX, and 1E1 IgG4. Representative fluorescence images showing the DQ signal and Hoechst nuclear staining. For each image, there is a black and white DQ fluorescence image converted at fixed settings. A graph showing the mean ± SEM of the calculated normalized degraded area per cell in several wells is shown with statistical significance (**) relative to the IgG control. [Figure 23] Collagen DQ degradation assay with 3T3 v-Src cells seeded for 48 hours in the presence of 20, 2, or 0.2 μg / ml IgG control, 1E1, 4G9, 1D6, and 2G9 IgG1. Representative fluorescence images showing the DQ signal and Hoechst nuclear staining. For each image, there is a black-and-white DQ fluorescence image converted at fixed settings. Graphs showing the mean ± SEM of the calculated normalized degraded area per cell in several wells are shown with statistical significance relative to the IgG control (* indicates p≦0.05, ** indicates p≦0.01, *** indicates p≦0.001, and **** indicates p≦0.0001). [Figure 24]Fluorescent gelatin layering assay with Huh7 cells seeded for 48 hours in the presence of 20, 2, or 0.2 μg / ml IgG control or the indicated IgG4. Representative fluorescent gelatin areas and accompanying Hoechst images are shown, along with a graph showing the mean ± SEM of the calculated normalized resolved area per cell in several fields for three biological replicates. [Figure 25] Images show in vivo visualization of liver tumor growth in various treatment groups on days 0 and 24. Quantitation of total photon emission from tumorigenic shp53 / Nras / luciferase-expressing liver tumors is shown in the upper right graph. Survival analysis of mice injected with CNX antibody compared to controls is shown in the lower right graph. [Figure 26] Survival analysis of a mouse model of breast cancer lung metastasis by tail vein injection of MDA-MB-231 ER-G2-tagged GFP cells supplemented with anti-CNX or control antibody is shown in the graph on the top right. Quantification of the number of nodules (mean ± standard deviation) in each condition is shown in the graph on the top right and visualized in the image below. [Figure 27] Comparison of subcutaneous tumor growth at day 10 in mice injected with NIH / 3T3vSrc (right flank) and NIN / 3T3vSrc CNX CALR KO (left flank) is shown in images (top) and graphs (bottom) taken on day 10. [Figure 28A] (A) Immunoblot analysis of human IgG showing significant accumulation of CNX antibody in 3T3vSrc tumor tissue compared to other tissues. (B) Histopathological analysis of NIH / 3T3vSrc tumors from mice treated with CNX antibody (middle column) or Ctrl IgG1 (left column) compared to NIH / 3T3vSrc CNX CALR KO tumors. (C) Co-stained immunohistofluorescence analysis of vimentin (fibroblast marker), human IgG1, and TUNEL (cell death) in NIH / 3T3vSrc tumor and liver samples from mice treated with α-CNX IgG1 or control IgG1. Scale bar: 500 μm. [Figure 28B](A) Immunoblot analysis of human IgG showing significant accumulation of CNX antibody in 3T3vSrc tumor tissue compared to other tissues. (B) Histopathological analysis of NIH / 3T3vSrc tumors from mice treated with CNX antibody (middle column) or Ctrl IgG1 (left column) compared to NIH / 3T3vSrc CNX CALR KO tumors. (C) Co-stained immunohistofluorescence analysis of vimentin (fibroblast marker), human IgG1, and TUNEL (cell death) in NIH / 3T3vSrc tumor and liver samples from mice treated with α-CNX IgG1 or control IgG1. Scale bar: 500 μm. [Figure 28C] (A) Immunoblot analysis of human IgG showing significant accumulation of CNX antibody in 3T3vSrc tumor tissue compared to other tissues. (B) Histopathological analysis of NIH / 3T3vSrc tumors from mice treated with CNX antibody (middle column) or Ctrl IgG1 (left column) compared to NIH / 3T3vSrc CNX CALR KO tumors. (C) Co-stained immunohistofluorescence analysis of vimentin (fibroblast marker), human IgG1, and TUNEL (cell death) in NIH / 3T3vSrc tumor and liver samples from mice treated with α-CNX IgG1 or control IgG1. Scale bar: 500 μm. [Figure 29] Effect of scFV clone 8 in the CAIA model. (A) Treatment schedule for collagen antibody-induced arthritis in a C57BL6 mouse model. Anti-collagen antibody was injected on day 0, followed by an LPS challenge injection on day 3. On days 3, 5, 7, and 9, scFV clone 8 was intraperitoneally injected at 100 μg per mouse. Control mice received PBS instead of scFV clone 8. Mice were sacrificed on day 10. (B) Variation in paw thickness was measured across various time points, and statistically significant differences are indicated (* indicates p≦0.05, ** indicates p≦0.01, and *** indicates p≦0.001). (C) Arthritis scores measured on days 7 and 10 for control and scFV-injected mouse groups. Statistically significant differences are indicated at day 10 (*). [Figure 30]Effect of IgG4 1E1 in the CAIA model. (A) Treatment schedule in the setting of collagen antibody-induced arthritis in a C57BL6 mouse model. Anti-collagen antibody was injected on day 0, followed by an LPS challenge injection on day 3, and IgG4 1E1 was injected intraperitoneally at 250 μg per mouse on days 3, 5, and 7. Control mice received PBS instead of IgG4 1E1. Mice were sacrificed on day 10. (B) Variation in paw thickness was measured across various time points, and statistically significant daily differences are indicated (* indicates p ≤ 0.05, ** indicates p ≤ 0.01, and *** indicates p ≤ 0.001). [Figure 31] O-glycosylation is enhanced in human samples with rheumatoid arthritis and osteoarthritis, indicating that high levels of O-glycosylation correlate with disease status. Panel A shows representative images of immunohistofluorescence staining of nuclei with Hoechst (upper panel) and O-GalNAc glycans (Tn glycans) stained with Vicia Villosa lectin (VVL, lower panel) for human tissue microarrays (TMAs) containing joint tissue from healthy subjects (normal) and patients with osteoarthritis (OA), rheumatoid arthritis (RA), or psoriatic arthritis (PSA). Scale bar: 5 μm. Panel B shows a quantitative graph of Tn glycan levels in individual tissue cores. Osteoarthritis patients exhibited higher O-GalNAc glycan levels than healthy subjects, while most rheumatoid arthritis patients and two psoriatic arthritis patients exhibited higher O-GalNAc glycan levels than healthy individuals. Data are means ± SEM and were obtained from two separate tissue microarray (TMA) slides consisting of tissue sections from 21 osteoarthritis patients, 18 rheumatoid arthritis patients, 6 psoriatic arthritis patients, and 7 healthy controls. Individual data points represent the raw integrated density of Vicia faba lectin (VVL) staining normalized to the density of nuclear staining in each individual subject. Box and whisker plots show all values, with boxes extending from the 25th to 75th percentiles and error bars ranging from maximum to minimum. *: p<0.05, ****: p<0.0001, NS: not significant (one-way ANOVA, Kruskal-Wallis test). [Figure 32-1] Tn glycan levels were elevated in mice with induced arthritis and correlated with disease severity, indicating that high Tn glycan levels correlate with disease status. Panel A shows hematoxylin and eosin (HE) histology results of synovial tissue from control mice (day 0) or mice with collagen type II antibody-induced arthritis (CAIA) at days 7, 10, and 14. S: synovium; B: bone; P: pannus; (*): immune cell infiltration in the underlying synovial lining; arrow: bone erosion. Panel B shows representative immunofluorescent staining images with Vicia faba lectin (VVL, upper panel) and nuclei (lower panel) demonstrating a time-dependent increase in VVL staining in pannus tissue from CAIA mice from days 7 to 10. Scale bar: 50 μm. Panels C and D show the results of clinical score assessment (C) and quantification of total Tn levels in the synovium (D) of CAIA mice from days 0 to 14. In panel C, data are the average arthritis scores of four mice per time point. In panel D, individual data points represent the average Tn levels of individual joints, calculated for two front paw joints of each animal. Box and whisker plots show all values, with boxes extending from the 25th to 75th percentiles and error bars ranging from maximum to minimum values. *: p<0.05, **: p<0.01, NS: not significant (one-way ANOVA). [Figure 32-2]Tn glycan levels were elevated in mice with induced arthritis and correlated with disease severity, indicating that high Tn glycan levels correlate with disease status. Panels C and D show the results of clinical score assessment (C) and quantification of total Tn levels in the synovium (D) of CAIA mice from days 0 to 14. In panel C, data are the average arthritis scores of four mice per time point. In panel D, individual data points represent the average Tn levels in individual joints, calculated for two front paw joints of each animal. Box and whisker plots show all values, with boxes extending from the 25th to 75th percentiles and error bars extending from the maximum to minimum values. *: p<0.05, **: p<0.01, NS: not significant (one-way ANOVA). [Figure 33-1] Synoviocytes from mice with collagen type II antibody-induced arthritis (CAIA) exhibit signs of GALA pathway activation. This indicates that the GALA pathway is active in synoviocytes in this disease model, making it a therapeutic target. Panels A and B show images of co-staining of Vicia faba lectin (VVL, A) or GALNT2 (B) with the endoplasmic reticulum (ER)-resident protein CNX (CNX), demonstrating significantly increased levels of Tn glycans in the synovium of mice with induced arthritis. Scale bar: 50 μm. Magnified images show VVL staining or GALNT2 enzyme co-localized with CNX in arthritic joints, indicating the activation state of GALA. Magnification scale: 4x; S: synovium; B: bone. Arrows indicate the Golgi (untreated mice) or ER staining pattern (CAIA mice) of VVL or GALNT2. [Figure 33-2]Synoviocytes from mice with collagen type II antibody-induced arthritis (CAIA) exhibit signs of GALA pathway activation. This indicates that the GALA pathway is active in synoviocytes in this disease model, making it a therapeutic target. Panels A and B show images of co-staining of Vicia faba lectin (VVL, A) or GALNT2 (B) with the endoplasmic reticulum (ER)-resident protein CNX (CNX), demonstrating significantly increased levels of Tn glycans in the synovium of mice with induced arthritis. Scale bar: 50 μm. Magnified images show VVL staining or GALNT2 enzyme co-localized with CNX in arthritic joints, indicating the activation state of GALA. Magnification scale: 4x; S: synovium; B: bone. Arrows indicate the Golgi (untreated mice) or ER staining pattern (CAIA mice) of VVL or GALNT2. [Figure 34] Synovial fibroblasts (SFs) are the predominant cell type exhibiting GALA activation in collagen type II antibody-induced arthritis (CAIA) mice, demonstrating that the GALA pathway is active in synovial cells in this disease model and thus represents a therapeutic target. Co-staining with Vicia faba lectin (lower right panel) and the fibroblast marker vimentin (lower left panel), the immune cell marker anti-CD45 (upper right panel), and nuclei (upper left panel) demonstrates the relative distribution of immune cells (circles) and fibroblasts (rectangles) and their Tn expression levels in the pannus tissue of CAIA mice. Scale bar: 50 μm. [Figure 35]Synovial lining fibroblasts from patients with osteoarthritis and rheumatoid arthritis exhibited strong GALA activation, indicating that the GALA pathway is active in synovial cells in disease models, thereby making it a therapeutic target. Panel A shows the results of hematoxylin and eosin (H&E) histology of synovial tissue from patients with rheumatoid arthritis and osteoarthritis. "*" indicates immune cell infiltration in the sublining layer of RA synovium. SL: synovial lining layer; scale bar: 100 μm. Panel B shows representative immunofluorescence images of osteoarthritis (upper panel) and rheumatoid arthritis (lower panel) synovium, demonstrating strong Tn glycan levels in synovial fibroblasts (SFs) of the lining, as identified by FAPα (arrowhead) and sparse Tn staining in immune cells identified by CD45 in the sublining layer (divided by dashed line). Scale bar: 50 μm. [Figure 36]Primary synovial fibroblasts (SFs) from patients with osteoarthritis and rheumatoid arthritis induce strong GALA activation in response to stimulation with arthritis-promoting cytokines and cartilage extracellular matrix (ECM). This indicates that the GALA pathway is involved in arthritic symptoms and disease progression. Panel A shows fluorescence-activated cell sorting (FACS) dot plots demonstrating highly pure (>90%) primary synovial fibroblast cultures established from healthy subjects (HCSFs), osteoarthritis (OASFs), and rheumatoid arthritis (RASFs). Panel B shows representative images of Helix pomatia lectin (HPL) staining showing higher Tn-glycan levels in osteoarthritic and rheumatoid arthritis synovial fibroblasts compared to HCSFs under basal conditions. The magnitude of Tn-glycan activation is accelerated after priming these cells with arthritis-promoting cytokines (cytokines), including IL-1β and TNFα, and cartilage extracellular matrix. Scale bar: 20 μm. Panel C shows the quantification of HPL levels in HCSFs, OASFs, and RASFs under basal conditions and after stimulation with CYTO alone (uncoated) or in combination with cartilage extracellular matrix or collagen type I extracellular matrix. Data are the mean ± SEM of two independent experiments. *p<0.05, ***<0.001, ****p<0.0001. NS: not significant (two-way ANOVA). [Figure 37-1]GALA activation in synovial fibroblasts promotes cartilage extracellular matrix degradation, indicating that the GALA pathway is involved in arthritis symptoms and disease progression. Panel A shows a strategy to inhibit GALA in synovial fibroblasts by stably transfecting SW982 synovial fibroblasts with a construct (ER-2Lec) expressing two lectin domains of GALNT2 in the ER, which are induced by doxycycline (DOX). Panel B shows representative images of the matrix degradation activity of SW982 cells and SW982 cells expressing ER-2Lec after stimulation with rheumatoid arthritis-associated cytokines (CYTO; IL1β and TNFα). Arrows indicate degraded matrix. Panel C shows a quantitative graph demonstrating the reduced matrix degradation activity in SW982 cells with GALA inhibition after stimulation with CYTO (IL1β and TNFα). Data correspond to the mean ± SEM and represent three independent experiments. Each data point represents the total resolved area (μm²) per nucleus per well. ***: p<0.001, ****: p<0.0001, One-way ANOVA). Figure includes SEQ ID NO: 408. [Figure 37-2]GALA activation in synovial fibroblasts promotes cartilage extracellular matrix degradation, indicating that the GALA pathway is involved in arthritis symptoms and disease progression. Panel A shows a strategy to inhibit GALA in synovial fibroblasts by stably transfecting SW982 synovial fibroblasts with a construct (ER-2Lec) expressing two lectin domains of GALNT2 in the ER, which are induced by doxycycline (DOX). Panel B shows representative images of the matrix degradation activity of SW982 cells and SW982 cells expressing ER-2Lec after stimulation with rheumatoid arthritis-associated cytokines (CYTO; IL1β and TNFα). Arrows indicate degraded matrix. Panel C shows a quantitative graph demonstrating the reduced matrix degradation activity in SW982 cells with GALA inhibition after stimulation with CYTO (IL1β and TNFα). Data correspond to the mean ± SEM and represent three independent experiments. Each data point represents the total resolved area (μm²) per nucleus per well. ***: p<0.001, ****: p<0.0001, One-way ANOVA). Figure includes SEQ ID NO: 408. [Figure 38] Expression of ER-2Lec in synovial fibroblasts in vivo suppresses GALA activation, demonstrating the efficacy of ER-2Lec expression in treating arthritis or alleviating arthritis-associated symptoms. Representative images are shown showing that ER-2Lec is primarily expressed in synovial fibroblasts, identified by EGFP-positive staining (arrowheads), in the synovium of Col6a1Cre ER-2Lec mice, and that such cells exhibit reduced VVL staining, indicating inhibition of GALA. B: Bone; S: Synovium; Scale bar: 50 μm. [Figure 39]Effect of GALA inhibition by ER-2Lec expression on reducing paw swelling in CAIA mice. This demonstrates the effectiveness of ER-2Lec expression in treating arthritis or alleviating arthritis-associated symptoms. Panel A shows representative images, and panel B shows quantification. Paw thickness analysis demonstrates reduced swelling levels in the front and hind paws of Col6a1Cre ER-2Lec mice 7 days after arthritis induction compared to Col6a1Cre control mice. Data are mean ± SEM of two independent experiments. n = 5 mice per group. **: p < 0.01 (one-way ANOVA). [Figure 40] Inhibition of GALA by expression of ER-2Lec in synovial fibroblasts (SF) reduced clinical scores in CAIA mice, demonstrating that expression of ER-2Lec is effective in treating arthritis on a clinical scale. Clinical score measurements showed that the severity of arthritis was reduced in Col6a1Cre ER-2Lec mice 7 days after induction of arthritis compared with Col6a1Cre control mice. Data are the mean ± SEM of two independent experiments. n = 5 mice per group. P = 0.09 (non-parametric t-test, Mann-Whitney test). [Figure 41-1]Inhibition of GALA by expression of ER-2Lec in synovial fibroblasts protected CAIA mice from cartilage degradation, demonstrating the effectiveness of ER-2Lec expression in treating arthritis or alleviating arthritis-associated symptoms. Panels A and B show representative images of Alcian blue (AB) staining (A) and Safranin-O (SO) staining (B) from untreated Col6a1Cre mice or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7. Panels C and D show quantification of the area of positive AB staining (C) and SO staining (D) (scale bar). The data showed that arthritis-induced cartilage matrix degradation was reversed in GALA-inhibited mice (Col6aCre ER-2Lec). Each data point represents the average area of positive staining per mm2 of articular cartilage from three different metacarpophalangeal joints from one animal. Data are shown as mean ± SEM. *: p<0.05, **: p<0.01, **: p<0.001 (one-way ANOVA test). [Figure 41-2] Inhibition of GALA by expression of ER-2Lec in synovial fibroblasts protected CAIA mice from cartilage degradation, demonstrating the effectiveness of ER-2Lec expression in treating arthritis or alleviating arthritis-associated symptoms. Panels A and B show representative images of Alcian blue (AB) staining (A) and Safranin-O (SO) staining (B) from untreated Col6a1Cre mice or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7. Panels C and D show quantification of the area of positive AB staining (C) and SO staining (D) (scale bar). The data showed that arthritis-induced cartilage matrix degradation was reversed in GALA-inhibited mice (Col6aCre ER-2Lec). Each data point represents the average area of positive staining per mm2 of articular cartilage from three different metacarpophalangeal joints from one animal. Data are shown as mean ± SEM. *: p<0.05, **: p<0.01, **: p<0.001 (one-way ANOVA test). [Figure 42]GALA activates the O-glycosylation of CNX (CNX) in arthritis-induced synovial fibroblasts. This demonstrates the role of the GALA pathway in disease states and identifies CNX as a therapeutic target. Panels A and B show the results of co-immunoprecipitation blots using VVL lectin (A), and the quantitative graph (B) shows that the level of O-GalNAc-glycosylated CNX (CNX) is enhanced in SW982 cells after stimulation with arthritis-associated cytokines (CYTO) and cartilage extracellular matrix (ECM), but is reduced in SW982 cells expressing ER-2Lec induced by doxycycline (DOX). Actin was used as a loading control. In panel B, data are shown as mean ± SEM and are representative of three independent experiments. *: p<0.05, **: p<0.01 (one-way ANOVA test). [Figure 43] GALA induces cell surface exposure of CNX (CNX) in arthritis-induced synovial fibroblasts. This demonstrates the role of the GALA pathway in disease states and identifies CNX as a therapeutic target. Panels A and B show flow cytometry histogram plots (A) and quantification graphs (B) demonstrating an increased percentage of synovial fibroblasts expressing surface CNX (CNX) after stimulation with arthritis-induced cytokines (CYTO) and joint extracellular matrix (ECM). Induction of cell surface CNX is reduced in SF cells expressing ER-2Lec. In B, data are shown as mean ± SEM and are representative of two independent experiments. **: p<0.01, ***: p<0.001 (one-way ANOVA test). [Figure 44-1]Surface exposure of CNX is enhanced in primary synovial fibroblasts derived from both osteoarthritis (OA) and rheumatoid arthritis (RA) patients, identifying CNX as a therapeutic target for the treatment of arthritis. Panels A and B show flow cytometry histogram plots (A) and quantification graphs (B) demonstrating a greater proportion of osteoarthritic synovial fibroblasts (OASFs) or rheumatoid arthritis periosteal fibroblasts (RASFs) positive for surface CNX compared with healthy control synovial fibroblasts (HCSFs) under basal conditions or stimulation with arthritis-promoted cytokines (CYTOs) and cartilage extracellular matrix (ECM). In panel B, data are shown as mean ± SEM and are representative of two independent experiments. *: p<0.05, ***: p<0.001 (one-way ANOVA test). [Figure 44-2] Surface exposure of CNX is enhanced in primary synovial fibroblasts derived from both osteoarthritis (OA) and rheumatoid arthritis (RA) patients, identifying CNX as a therapeutic target for the treatment of arthritis. Panels A and B show flow cytometry histogram plots (A) and quantification graphs (B) demonstrating a greater proportion of osteoarthritic synovial fibroblasts (OASFs) or rheumatoid arthritis periosteal fibroblasts (RASFs) positive for surface CNX compared with healthy control synovial fibroblasts (HCSFs) under basal conditions or stimulation with arthritis-promoted cytokines (CYTOs) and cartilage extracellular matrix (ECM). In panel B, data are shown as mean ± SEM and are representative of two independent experiments. *: p<0.05, ***: p<0.001 (one-way ANOVA test). [Figure 45]Disulfide bonds are abundant in the cartilage extracellular matrix (ECM). This identifies the CNX:PDIA3 complex (this is the complex's reducing effect on disulfide bonds) as a therapeutic target. Representative immunofluorescence staining images show staining of collagen fibers, including type III collagen (Col III), type I collagen (Col I), and fibronectin, in the cartilage extracellular matrix (ECM). Collagen disulfide bonds were chemically reduced using TCEP, which can be detected by staining with the OX133 antibody, or left untreated (UT). Scale bar: 5 μm. [Figure 46] Blockade of CNX (CNX) reduces the cartilage-degrading activity of primary synovial fibroblasts from osteoarthritis patients, identifying CNX as a therapeutic target for the treatment of arthritis. Panels A and B show representative images (A) and quantitative graphs (B) demonstrating reduced matrix-degrading activity in primary fibroblasts isolated from osteoarthritis patients after incubation with anti-CNX or an isotype control antibody. Data represent the mean ± SEM and representative data from three independent experiments. Each data point represents the total area degraded (µm²) per nucleus per well. ***: p<0.001 (one-way ANOVA). [Figure 47] Treatment with antibodies against CNX (CNX) reduced paw swelling in CAIA mice, demonstrating the effectiveness of using anti-CNX antibodies in treating arthritis. Panel A shows a schematic diagram of the antibody treatment scheme. Panel B shows representative photographs of CAIA mice treated with anti-CNX antibodies or left untreated at day 10. Panel C shows a line graph plotting paw thickness measurements, demonstrating reduced paw swelling in CAIA animals after injection with anti-CNX antibodies. Data represent the mean ± SEM. n = 4-5 mice per group. *: p < 0.05 (two-way ANOVA test). [Figure 48]Treatment with antibodies against CNX (CNX) reduces the severity of arthritis in CAIA mice, demonstrating the effectiveness of using anti-CNX antibodies in treating arthritis. Clinical scores of CAIA mice 10 days after treatment with an isotype control antibody or anti-CNX antibodies. Data are mean ± SEM. n = 4-5 mice per group. P values are estimated by non-parametric t-test (Mann-Whitney test). [Figure 49-1] Treatment with antibodies against CNX (CNX) protects CAIA mice from cartilage degradation, demonstrating the effectiveness of anti-CNX antibodies in treating arthritis or preventing the worsening of arthritis or arthritis symptoms. Panels A and B show representative histological images showing Alcian blue (AB) staining (A) and Safranin-O (SO) staining (B) (arrowed bars) in CAIA mice treated with an isotype control or anti-CNX antibody. Panels C and D show quantitative graphs demonstrating increased AB and SO staining areas in CAIA mice treated with anti-CNX antibody compared to the group treated with an isotype control antibody. Individual data points represent the average positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal. n = 4–5 mice per group. Data are expressed as mean ± SEM. *: p < 0.05, **: p < 0.01 (Mann-Whitney test). [Figure 49-2]Treatment with antibodies against CNX (CNX) protects CAIA mice from cartilage degradation, demonstrating the effectiveness of anti-CNX antibodies in treating arthritis or preventing the worsening of arthritis or arthritis symptoms. Panels A and B show representative histological images showing Alcian blue (AB) staining (A) and Safranin-O (SO) staining (B) (arrowed bars) in CAIA mice treated with an isotype control or anti-CNX antibody. Panels C and D show quantitative graphs demonstrating increased AB and SO staining areas in CAIA mice treated with anti-CNX antibody compared to the group treated with an isotype control antibody. Individual data points represent the average positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal. n = 4–5 mice per group. Data are expressed as mean ± SEM. *: p < 0.05, **: p < 0.01 (Mann-Whitney test). [Figure 50] Anti-CNX (CNX) antibodies accumulate in the synovium of CAIA mice. Representative immunofluorescence images show co-staining of VVL and anti-CNX antibodies (arrowheads) on day 10 in CAIA mice injected with anti-CNX or isotype control antibodies. This demonstrates the accumulation of anti-CNX antibodies, demonstrating their ability to bind and target the synovium of CAIA mice, while in contrast, there is no binding with the control isotype antibody. This indicates that CNX is expressed by cells in the synovium of CAIA mice and that cells in the synovium can be specifically targeted by anti-CNX antibodies for therapeutic use. B: Bone; S: Synovium. Scale bar: 50 μm. [Figure 51-1]Screening results for GALA targets and validation of their effects on primary arthritic synovial fibroblasts. Panel A shows a schematic diagram of a high-content screen to select GALA targets and their blockade. Synovial fibroblast (SF) cells (SW982) were cultured on quenched fluorescent cartilage matrix components (DQ-collagen) in the presence of GALA target blockers (antibodies or siRNAs). GALA degradative activity in synovial fibroblasts resulted in an increased fluorescent signal (control panel), while blocker-treated synovial fibroblasts were able to inhibit degradative activity. Panel B shows the results of staining with VVL, demonstrating high expression of Tn glycans outside the Golgi (as identified by the Golgi marker dianthin), indicating GALA activation in primary synovial fibroblasts (OASF) isolated from osteoarthritis patients. Scale bar: 5 μm. Panel C shows a quantitative graph demonstrating reduced matrix-degrading activity in primary synovial fibroblasts (OASFs) isolated from osteoarthritis patients and treated with anti-CNX and anti-MMP14 antibodies. Each data point represents the ratio of raw integrated density of degraded DQ-collagen to nuclei per field. Data are mean ± SEM. n = 20 fields per group. *: p < 0.05, **: p < 0.01, NS: not significant (one-way ANOVA test). [Figure 51-2]Screening results for GALA targets and validation of their effects on primary arthritic synovial fibroblasts. Panel A shows a schematic diagram of a high-content screen to select GALA targets and their blockade. Synovial fibroblast (SF) cells (SW982) were cultured on quenched fluorescent cartilage matrix components (DQ-collagen) in the presence of GALA target blockers (antibodies or siRNAs). GALA degradative activity in synovial fibroblasts resulted in an increased fluorescent signal (control panel), while blocker-treated synovial fibroblasts were able to inhibit degradative activity. Panel B shows the results of staining with VVL, demonstrating high expression of Tn glycans outside the Golgi (as identified by the Golgi marker dianthin), indicating GALA activation in primary synovial fibroblasts (OASF) isolated from osteoarthritis patients. Scale bar: 5 μm. Panel C shows a quantitative graph demonstrating reduced matrix-degrading activity in primary synovial fibroblasts (OASFs) isolated from osteoarthritis patients and treated with anti-CNX and anti-MMP14 antibodies. Each data point represents the ratio of raw integrated density of degraded DQ-collagen to nuclei per field. Data are mean ± SEM. n = 20 fields per group. *: p < 0.05, **: p < 0.01, NS: not significant (one-way ANOVA test). [Figure 52] Characterization of mice injected with anti-CNX or isotype control antibodies. The data show comparable weight changes between the two. A: Weight changes in CAIA mice treated with isotype control or anti-CNX antibodies. B and C: Representative histological images and quantification of Safranin-O (SO) staining area (B) in CAIA mice treated with isotype control or anti-CNX antibodies. Individual data points represent the average positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal. n = 4–5 mice per group. (C) Data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01 (Mann-Whitney test). [Figure 53]Histological analysis of O-GalNAc(Tn) glycans in synovial tissue from arthritic patients and animals with induced arthritis. A: Representative immunohistofluorescence staining of O-GalNAc glycans with VVL lectin on human tissue microarray (TMA) sections from osteoarthritis (OA), psoriasis (PSA), rheumatoid arthritis (RA), and healthy subjects (normal). Magnification: 10x, Scale bar: 500 μm. B: HE histology (upper panel) and immunohistochemical staining with VVL lectin (lower panel) of mice with collagen type II antibody-induced arthritis (CAIA) at day 7 or untreated (UNT). [Figure 54] Quantification of OA synovial tissue analysis and purification of primary synovial fibroblasts. A: (Left) H&E histology of synovial tissue obtained from an OA patient. Scale bar: 100 μm; SL: synovial lining layer. (Right) Representative immunofluorescence images of OA synovium stained with VVL / CD45 and FAPα. Lining synovial fibroblasts are identified by FAPα (arrowheads). Immune cells are identified by CD45. The boundary between the sublining layer and the lining layer is defined by a dashed line. Scale bar: 50 μm. B: FACS dot plots showing highly pure (>90%) primary SF cultures established from healthy subjects (HCSF), OA (OASF), and RA (RASF) patients. [Figure 55] GALA activation induces cartilage damage in CAIA mice. A: Representative images of safranin-O (SO) staining (B) from untreated Col6a1Cre mice or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7. Scale bar: 100 μm. B and C: Quantification of SO staining thickness (B) and total positive staining area (C). Arthritis-induced cartilage matrix degradation is indicated by arrowheads. Each data point represents the average positive staining area per mm2 of articular cartilage from three different metacarpophalangeal joints from one animal. Data are shown as mean ± SEM. *p<0.01, ****: p<0.0001 (one-way ANOVA test). [Figure 56]Binding data for monoclonal anti-CNX antibody clone 2G9. Data were generated using an ELISA assay. The assay revealed high specificity for CNX (left bar) and low binding to BSA-coated wells (right bar) of clone 2G9 and the commercial polyclonal antibody ab10286. The negative control hIgG did not significantly bind to CNX. Data represent the mean ± SEM and representative data from three independent experiments. [Figure 57] Binding data for monoclonal anti-CNX antibody clone 2G9 based on ELISA assays performed with serial dilutions of antibodies 2G9 and ab10286. The data suggest higher affinity and / or avidity of 2G9 compared to the commercially available polyclonal antibody ab10286. [Figure 58] ECM degradation assay data demonstrating the ECM degradation ability of monoclonal anti-CNX antibody clone 2G9. Panels A and B show representative images (A) and a quantitative graph (B) demonstrating that 2G9 can reduce ECM degradation by 90% compared to untreated controls. Data represent the mean ± SEM and representative data from three independent experiments. [Figure 59] Data from ECM degradation assay. 2G9 in IgG4 format can also block ECM degradation. Data represent the mean ± SEM and representative data from three independent experiments. ***: p<0.001 (One-way ANOVA). [Figure 60A]The effect of using IgG1 anti-CNX antibody on the expansion of Huh7 spheroids in Matrigel. (A) Representative reconstructed brightfield images of 96-well plates containing Huh7 spheroids on days 1 and 12 after the indicated treatments. The solid line represents the spheroid area on day 1. The dashed line represents the same spheroid area as on day 1, but shows an expansion in size on day 12. The dashed line represents the same spheroid area as on day 1, but shows a decrease in size on day 12. IgG1 was used at 10 μg / ml. (B) Line plots of spheroid size change on days 1 and 12 for the indicated treatments. Each line depicts a spheroid monitored in the same well and image location on days 1 and 12. (C) Violin and box plots of spheroid growth on day 12 relative to day 1. P values indicate significant differences in spheroid growth in the 1E1-treated condition relative to the control IgG1. Day 1 was set as 100%. [Figure 60B] The effect of using IgG1 anti-CNX antibody on the expansion of Huh7 spheroids in Matrigel. (A) Representative reconstructed brightfield images of 96-well plates containing Huh7 spheroids on days 1 and 12 after the indicated treatments. The solid line represents the spheroid area on day 1. The dashed line represents the same spheroid area as on day 1, but shows an expansion in size on day 12. The dashed line represents the same spheroid area as on day 1, but shows a decrease in size on day 12. IgG1 was used at 10 μg / ml. (B) Line plots of spheroid size change on days 1 and 12 for the indicated treatments. Each line depicts a spheroid monitored in the same well and image location on days 1 and 12. (C) Violin and box plots of spheroid growth on day 12 relative to day 1. P values indicate significant differences in spheroid growth in the 1E1-treated condition relative to the control IgG1. Day 1 was set as 100%. [Figure 60C]The effect of using IgG1 anti-CNX antibody on the expansion of Huh7 spheroids in Matrigel. (A) Representative reconstructed brightfield images of 96-well plates containing Huh7 spheroids on days 1 and 12 after the indicated treatments. The solid line represents the spheroid area on day 1. The dashed line represents the same spheroid area as on day 1, but shows an expansion in size on day 12. The dashed line represents the same spheroid area as on day 1, but shows a decrease in size on day 12. IgG1 was used at 10 μg / ml. (B) Line plots of spheroid size change on days 1 and 12 for the indicated treatments. Each line depicts a spheroid monitored in the same well and image location on days 1 and 12. (C) Violin and box plots of spheroid growth on day 12 relative to day 1. P values indicate significant differences in spheroid growth in the 1E1-treated condition relative to the control IgG1. Day 1 was set as 100%. [Figure 61A] (A) Schematic of the fluorescent gelatin ECM degradation assay. Degraded fluorescent gelatin on the coverslip appears as a dark shadow, and the degraded area was quantified. (B) Representative images of the degraded area of gelatin and nuclei with various antibody treatments. Dox- refers to SW982 ERG1 cells not induced with doxycycline, while Dox+ refers to cells induced with 1 μg / ml of doxycycline. All cells treated with 10 μg / ml of antibody were induced with doxycycline. Ctl hIgG1 corresponds to SW982 cells treated with irrelevant human IgG1. ab22595 and ab92573 are commercially available antibodies targeting CNX. Two experimental replicates were performed. (C) Quantification of the degraded ECM area per nucleus in the presence of various antibody treatments. Each point represents one imaging field. Results are a combination of two experimental replicates. [Figure 61B](A) Schematic of the fluorescent gelatin ECM degradation assay. Degraded fluorescent gelatin on the coverslip appears as a dark shadow, and the degraded area was quantified. (B) Representative images of the degraded area of gelatin and nuclei with various antibody treatments. Dox- refers to SW982 ERG1 cells not induced with doxycycline, while Dox+ refers to cells induced with 1 μg / ml of doxycycline. All cells treated with 10 μg / ml of antibody were induced with doxycycline. Ctl hIgG1 corresponds to SW982 cells treated with irrelevant human IgG1. ab22595 and ab92573 are commercially available antibodies targeting CNX. Two experimental replicates were performed. (C) Quantification of the degraded ECM area per nucleus in the presence of various antibody treatments. Each point represents one imaging field. Results are a combination of two experimental replicates. [Figure 61C] (A) Schematic of the fluorescent gelatin ECM degradation assay. Degraded fluorescent gelatin on the coverslip appears as a dark shadow, and the degraded area was quantified. (B) Representative images of the degraded area of gelatin and nuclei with various antibody treatments. Dox- refers to SW982 ERG1 cells not induced with doxycycline, while Dox+ refers to cells induced with 1 μg / ml of doxycycline. All cells treated with 10 μg / ml of antibody were induced with doxycycline. Ctl hIgG1 corresponds to SW982 cells treated with irrelevant human IgG1. ab22595 and ab92573 are commercially available antibodies targeting CNX. Two experimental replicates were performed. (C) Quantification of the degraded ECM area per nucleus in the presence of various antibody treatments. Each point represents one imaging field. Results are a combination of two experimental replicates. [Figure 62] (A) Paw thickness variation from day 0 to day 17 in CAIA mice treated with 1E1 or control PBS. (B) Unpaired t test demonstrates significant differences in paw thickness variation from day 0 to day 17 in control and 1E1-treated mice. [Figure 63](A) Variation in paw thickness from day 0 to day 17 in CAIA mice treated with 2G9 or control IgG1. (B) Unpaired t-test demonstrates significant differences in the variation in paw thickness from day 0 to day 17 in control and 2G9-treated mice. [Figure 64A] (A) Representative images of the area of gelatin and nuclei degraded by HUH7 cells (top) and SW982 ERG1 cells (bottom). Cells were treated with various antibodies at doses of 2 μg / ml, 1 μg / ml, and 0.2 μg / ml for two days. SW982 ERG1 cells were induced to activate GALA with 1 μg / ml doxycycline. Ctl hIgG1 corresponds to cells treated with irrelevant human IgG1. ab22595 is a commercially available antibody targeting CNX. (B) Quantification of the area of ECM degraded per nucleus in HUH7 (left) and SW982 ERG1 (right) cells treated with various antibodies at doses of 2 μg / ml, 1 μg / ml, and 0.2 μg / ml for two days. Each point represents one imaging field. [Figure 64B] (A) Representative images of the area of gelatin and nuclei degraded by HUH7 cells (top) and SW982 ERG1 cells (bottom). Cells were treated with various antibodies at doses of 2 μg / ml, 1 μg / ml, and 0.2 μg / ml for two days. SW982 ERG1 cells were induced to activate GALA with 1 μg / ml doxycycline. Ctl hIgG1 corresponds to cells treated with irrelevant human IgG1. ab22595 is a commercially available antibody targeting CNX. (B) Quantification of the area of ECM degraded per nucleus in HUH7 (left) and SW982 ERG1 (right) cells treated with various antibodies at doses of 2 μg / ml, 1 μg / ml, and 0.2 μg / ml for two days. Each point represents one imaging field. [Figure 65] Immunoblot analysis of human IgG1 showed that the CNX antibody significantly accumulated in HepG2-Luc tumor tissue compared with the control. [Figure 66]Immunoblot analysis of human IgG1 showed that the CNX antibody significantly accumulated in Hep3B tumor tissue compared with the control. [Figure 67] Immunoblot analysis of human IgG1 showed that the CNX antibody significantly accumulated in Huh7-Luc tumor tissue compared with the control. [Figure 68A] Determination of the amount of CNX antibody, designated 1E1, accumulated in various tumors (HepG2Luc, Hep3B, and Huh7Luc) from NSG mice treated with 1E1 or control IgG1 (EBOLA) at three doses of 30 mg / kg every two days. (A) Western blot of 25 μg / well of tumor with serial dilutions of IgG1. (B) Standard curve generated by ImageJ analysis using Western blot intensities of heavy and light chains normalized to background. (C) Amount of IgG1 in 1 μg of tumor tissue (mean ± SD). [Figure 68B] Determination of the amount of CNX antibody, designated 1E1, accumulated in various tumors (HepG2Luc, Hep3B, and Huh7Luc) from NSG mice treated with 1E1 or control IgG1 (EBOLA) at three doses of 30 mg / kg every two days. (A) Western blot of 25 μg / well of tumor with serial dilutions of IgG1. (B) Standard curve generated by ImageJ analysis using Western blot intensities of heavy and light chains normalized to background. (C) Amount of IgG1 in 1 μg of tumor tissue (mean ± SD). [Figure 68C] Determination of the amount of CNX antibody, designated 1E1, accumulated in various tumors (HepG2Luc, Hep3B, and Huh7Luc) from NSG mice treated with 1E1 or control IgG1 (EBOLA) at three doses of 30 mg / kg every two days. (A) Western blot of 25 μg / well of tumor with serial dilutions of IgG1. (B) Standard curve generated by ImageJ analysis using Western blot intensities of heavy and light chains normalized to background. (C) Amount of IgG1 in 1 μg of tumor tissue (mean ± SD). [Figure 69A](A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 7, and 20. Tumor-bearing nude mice were treated with CNX antibody (1E1) at a dose of 15 mg / kg compared to the IgG1 control. (B) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors. (C) Quantification of total photon emission from luciferase-expressing Huh7Luc tumors. Mean ± SD. [Figure 69B] (A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 7, and 20. Tumor-bearing nude mice were treated with CNX antibody (1E1) at a dose of 15 mg / kg compared to the IgG1 control. (B) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors. (C) Quantification of total photon emission from luciferase-expressing Huh7Luc tumors. Mean ± SD. [Figure 69C] (A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 7, and 20. Tumor-bearing nude mice were treated with CNX antibody (1E1) at a dose of 15 mg / kg compared to the IgG1 control. (B) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors. (C) Quantification of total photon emission from luciferase-expressing Huh7Luc tumors. Mean ± SD. [Figure 70A] (A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 18, and 27. Tumor-bearing NSG mice were treated with CNX antibodies (1E1 or 3D1) compared to IgG1 control at a dose of 15 mg / kg. (B) Quantification of total photon emission from HepG2Luc tumors (1 million cells). (C) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors (5 million cells). Mean ± SD. [Figure 70B](A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 18, and 27. Tumor-bearing NSG mice were treated with CNX antibodies (1E1 or 3D1) compared to IgG1 control at a dose of 15 mg / kg. (B) Quantification of total photon emission from HepG2Luc tumors (1 million cells). (C) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors (5 million cells). Mean ± SD. [Figure 70C] (A) Images show in vivo visualization of subcutaneous tumor growth in various treatment groups on days 0, 18, and 27. Tumor-bearing NSG mice were treated with CNX antibodies (1E1 or 3D1) compared to IgG1 control at a dose of 15 mg / kg. (B) Quantification of total photon emission from HepG2Luc tumors (1 million cells). (C) Quantification of total photon emission from luciferase-expressing HepG2Luc tumors (5 million cells). Mean ± SD. [Figure 71A] (A) Schematic diagram of in vivo imaging experiment. (B) In vivo bioluminescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (C) In vivo fluorescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (D) Ex vivo imaging of calnexin / 1E1 Alexa Fluor® 680 conjugate accumulation in tumors and organs 5 days after injection. [Figure 71B] (A) Schematic diagram of in vivo imaging experiment. (B) In vivo bioluminescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (C) In vivo fluorescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (D) Ex vivo imaging of calnexin / 1E1 Alexa Fluor® 680 conjugate accumulation in tumors and organs 5 days after injection. [Figure 71C] (A) Schematic diagram of in vivo imaging experiment. (B) In vivo bioluminescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (C) In vivo fluorescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (D) Ex vivo imaging of calnexin / 1E1 Alexa Fluor® 680 conjugate accumulation in tumors and organs 5 days after injection. [Figure 71D] (A) Schematic diagram of in vivo imaging experiment. (B) In vivo bioluminescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (C) In vivo fluorescence imaging of α-calnexin / 1E1 Alexa Fluor® 680 conjugate in mice bearing HepG2Luc tumors. (D) Ex vivo imaging of calnexin / 1E1 Alexa Fluor® 680 conjugate accumulation in tumors and organs 5 days after injection. DETAILED DESCRIPTION OF THE INVENTION

[0027] explanation The present disclosure provides antigen-binding molecules that bind to CNX and have novel biophysical and / or functional properties compared to antigen-binding molecules disclosed in the prior art. CNX and CRT The present disclosure relates to CNX-specific antigen-binding molecules.

[0028] Human CNX (also known as CNX, CANX, or IP90) is a protein identified by UniProt P27824. Alternative splicing of the mRNA encoded by the human CANX gene results in three major CNX isoforms: isoform 1 (SEQ ID NO: 333), isoform 2 (SEQ ID NO: 334), and isoform 3 (SEQ ID NO: 335). Isoform 2 differs from isoform 1 by the insertion of a 35 amino acid sequence after position 1 of SEQ ID NO: 333. Positions 1-108 of SEQ ID NO: 333 are absent in isoform 3.

[0029] Human CNX isoform 1 contains an N-terminal signal peptide (SEQ ID NO: 336), followed by a calcium-binding luminal domain (SEQ ID NO: 337), a single-pass transmembrane domain (SEQ ID NO: 338), and a C-terminal acidic cytoplasmic domain (SEQ ID NO: 339). The luminal domain contains a globular lectin domain (SEQ ID NO: 340), followed by an arm-like proline-rich P domain (SEQ ID NO: 341), and a second lectin domain (SEQ ID NO: 342). The mature form of human CNX isoform 1 is set forth in SEQ ID NO: 343.

[0030] As used herein, "CNX" refers to CNX derived from any species, including isoforms, fragments, variants, or homologs derived from any species. In some embodiments, the CNX is derived from a mammal (e.g., a Therian, a Placental, a Supertheria, a Microtheria, an Archonta, a Primate (e.g., a rhesus monkey, a cynomolgus monkey, a non-human primate, or a human). In some embodiments, the CNX is a human CNX or a mouse CNX.

[0031] As used herein, a "fragment," "variant," "isoform," or "homologue" of a given protein is optionally characterized as having at least 60%, preferably one or more of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with the amino acid sequence of a reference protein (e.g., a reference isoform).

[0032] A "fragment" generally refers to a fraction of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein (e.g., human CNX isoform 1, isoform 2, and isoform 3 are all isoforms of each other). A "homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. For example, human CNX isoform 1 (UniProt: P27824-1, v2; SEQ ID NO: 333) and mouse CNX (UniProt: P35564-1, v1; SEQ ID NO: 344) are homologs of each other. Homologs include orthologs.

[0033] Isoforms, fragments, variants, or homologs of CNX according to the present disclosure are characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature CNX isoform, optionally from a given species, e.g., human.

[0034] An isoform, fragment, variant, or homolog may optionally be a functional isoform, fragment, variant, or homolog that has, for example, a functional property / activity of a reference CNX (e.g., human CNX isoform 1), as determined by analysis with a suitable assay for functional property / activity. For example, an isoform, fragment, variant, or homolog of CNX may exhibit binding to N-glycoproteins bearing monoglycosylated N-glycans and / or association with ERp57, cyclophilin B, and / or ERp29.

[0035] In some embodiments, CNX comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 333, 334, 335, or 343.

[0036] In some embodiments, CNX comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 344 or 352.

[0037] A "fragment" of a reference protein may be of any length (in terms of number of amino acids), but may optionally be at least 25% of the length of the reference protein (i.e., the protein from which the fragment is derived), and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0038] A fragment of CNX may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acids, and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acids.

[0039] In some embodiments, a fragment of CNX comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 343, 337, 338, 339, 340, 341, or 342.

[0040] In some embodiments, a fragment of CNX comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 352, 346, 347, 348, 349, 350, or 351.

[0041] In some aspects, the antigen-binding molecules of the present disclosure exhibit binding to calreticulin (CRT). We observed that CNX-depleted cells could compensate for the loss of CNX through the action of CRT, and therefore identified an antigen-binding molecule that could bind to both CNX and CRT.

[0042] In some embodiments, the antigen-binding molecule is cross-reactive with human CNX and CRT. In some embodiments, the antigen-binding molecule reduces the activity of CNX and CRT. In some embodiments, the antigen-binding molecule reduces the activity of CNX and CRT.

[0043] As used herein, a "cross-reactive" antigen-binding molecule / domain binds to the target antigen with which the antigen-binding molecule / domain is cross-reactive.For example, an antigen-binding molecule / domain / polypeptide that is cross-reactive with CNX and CRT can bind to CNX and CRT.A cross-reactive antigen-binding molecule / domain / polypeptide can exhibit specific binding to each of the target antigens.

[0044] Human CRT (also known as calreticulin, calregulin, or ERp60) is a protein identified by UniProt P27797. Human CRT has the amino acid sequence set forth in SEQ ID NO: 353. Human CRT contains an N-terminal signal peptide (SEQ ID NO: 354) followed by a calcium-binding N-domain (SEQ ID NO: 355), and a C-terminal acidic C-domain (SEQ ID NO: 356). The N-domain contains a globular lectin domain (SEQ ID NO: 357) followed by an arm-like proline-rich P-domain (SEQ ID NO: 359), and a second lectin domain (SEQ ID NO: 358). The mature form of human CRT is set forth in SEQ ID NO: 360.

[0045] As used herein, "CRT" refers to CRT from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, the CRT is from a mammal (e.g., a Therian, a Placental, a Supertheria, a Microtheria, an Archonta, a Primate (e.g., a rhesus monkey, a cynomolgus monkey, a non-human primate, or a human). In some embodiments, the CRT is a human CRT or a mouse CRT.

[0046] Isoforms, fragments, variants, or homologs of CRT according to the present disclosure are characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature CRT isoform, optionally from a given species, e.g., human.

[0047] An isoform, fragment, variant, or homolog of CRT can optionally be a functional isoform, fragment, variant, or homolog that has, for example, a functional property / activity of a reference CRT (e.g., human CRT), as determined by analysis with a suitable assay for the functional property / activity. For example, an isoform, fragment, variant, or homolog of CRT can exhibit binding to N-glycoproteins bearing monoglycosylated N-glycans and / or association with ERp57, cyclophilin B, and / or ERp29.

[0048] In some embodiments, the CRT comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 353 or 360.

[0049] A fragment of CRT may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0050] In some embodiments, a fragment of CRT comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 360, 355, 356, 357, 358, or 359.

[0051] The structure and function of CNX and CRT are reviewed, for example, in Kozlov and Gehring, FEBS J. (2020) 287(20):4322-4340, the entire contents of which are incorporated herein by reference.

[0052] CNX and CRT are lectin chaperone proteins resident in the endoplasmic reticulum (ER). CNX / CRT binds to N-glycoproteins carrying monoglycosylated glycans and recruits various other chaperones that mediate protein disulfide formation, proline isomerization, and protein folding. CNX / CRT can associate with the protein folding enzyme ERp57 to catalyze glycoprotein-specific disulfide bond formation. The CNX:ERp57 complex has also been shown to translocate to the surface of cancer cells, where it reduces disulfide bridges in the extracellular matrix (Ros et al., Nat. Cell Biol. 22, pp. 1371–1381, 2020). Disulfide bridge reduction has been shown to be essential for the effective activity of matrix metalloproteinases (MMPs) and therefore extracellular matrix degradation in cancer. CNX / CRT also associates with cyclophilin B (CypB), a peptidyl-prolyl cis-trans isomerase, for proline isomerization of peptide bonds. CNX / CRT has also been reported to associate with ERp29 to form the CNX / CRT:ERp29 complex, which has general chaperone function. CNX also functions as a chaperone for the folding of the MHC class I α chain in the ER membrane.

[0053] Processing by glucosidase II removes the glucose residues from monoglycosylated N-glycans, which are necessary for the interaction between glycoproteins and CNX / CRT, thereby releasing the mature, processed glycoprotein from CNX / CRT. For improperly folded proteins, UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by re-adding glucose residues to the N-glycan, reconstituting the interaction site for CNX / CRT. In this way, misfolded proteins reassociate with CNX / CRT for further rounds of chaperone-mediated refolding, preventing them from exiting the ER and proceeding to the Golgi apparatus. If multiple folding cycles are unsuccessful, the misfolded protein is eventually transported to the cytoplasm and degraded via the ER-associated protein degradation (ERAD) pathway. antigen binding molecule The present disclosure provides antigen-binding molecules capable of binding to CNX. An antigen-binding molecule capable of binding to a given target antigen may be expressed as an antigen-binding molecule that binds to the given target antigen.

[0054] An "antigen-binding molecule" refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Ig)) and antigen-binding fragments thereof. As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antigen-binding molecules derived from antibodies, such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH), etc. Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab')2, and F(ab')2 fragments. In some embodiments, the antigen-binding molecule may be an antibody or an antigen-binding fragment thereof.

[0055] Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, e.g., molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules include an antigen-binding region / domain that comprises or consists of the antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g., provided as an scFv) or Fab region of an antibody, or a whole antibody. For example, antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) comprising a (cytotoxic) drug moiety (e.g., as described below). Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules, such as immune cell engager molecules that contain domains for recruiting (effector) immune cells, including BiTE, BiKE, and TriKE (reviewed, e.g., in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418-434 and Ellerman, Methods (2019) 154:102-117, both of which are incorporated herein by reference in their entirety). Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors that provide both antigen-binding and T-cell activation functions (the structure, function, and engineering of CARs are reviewed in Dotti et al., Immunol Rev (2014) 257(1), which is incorporated herein by reference in its entirety).

[0056] The antigen-binding molecules of the present disclosure comprise a portion capable of binding to a target antigen. In some embodiments, the portion capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to the target antigen. In some embodiments, the portion capable of binding to a target antigen comprises or consists of an aptamer, such as a nucleic acid aptamer, capable of binding to the target antigen (e.g., as reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin, as reviewed in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85, and Emanuel et al., Mabs (2011) 3:38-48).

[0057] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically range in length from about 2 to 50 amino acids. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0058] The antigen-binding molecules of the present disclosure generally comprise an antigen-binding domain comprising the VH and VL of an antibody capable of specifically binding to a target antigen. The antigen-binding domain formed by the VH and VL is also referred to herein as the Fv region.

[0059] An antigen-binding molecule may be or comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex. An antigen-binding molecule may comprise two or more polypeptides that together form an antigen-binding domain. The polypeptides may be associated by covalent or non-covalent bonds. In some embodiments, a polypeptide forms part of a larger polypeptide that comprises the polypeptide (e.g., in the case of an scFv comprising a VH and a VL, or in the case of an scFab comprising a VH-CH1 and a VL-CL).

[0060] An antigen-binding molecule may also refer to a non-covalent or covalent complex of two or more polypeptides (e.g., two, three, four, six, or eight polypeptides), such as an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0061] The antigen-binding molecules of the present disclosure may be designed and prepared using the sequence of a monoclonal antibody (mAb) capable of binding to CNX. Antigen-binding regions of antibodies, such as single-chain variable fragments (scFv), Fab, and F(ab')2 fragments, may also be used / provided. An "antigen-binding region" is any fragment of an antibody that binds to a target for which a given antibody is specific.

[0062] Antibodies generally contain six complementarity determining region (CDRs): three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the antibody paratope, which is the part of the antibody that binds to the target antigen.

[0063] The VH and VL regions comprise framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N- to C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0064] There are several different conventions for defining antibody CDRs and FRs, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, described in Retter et al., Nucl. Acids Res. (2005) 33(suppl 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43(Database issue):D413-22). This uses the IMGT V-DOMAIN numbering rules described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In a preferred embodiment, the CDRs and FRs of the antigen-binding molecules referred to herein are defined according to the IMGT information system.

[0065] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule that binds to CNX. That is, in some embodiments, the antigen-binding molecule comprises the VH region and VL region of an antigen-binding molecule that binds to CNX.

[0066] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of a CNX-binding antibody clone described herein, or the CDRs, FRs, and / or VH and / or VL regions derived therefrom of a CNX-binding antibody clone described herein. In some embodiments, the CNX-binding antibody clone is selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046, and C117.

[0067] In some embodiments, the antigen-binding molecule is selected from the following (1) to (19): (1) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 4 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (2) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 18; HC-CDR2 having the amino acid sequence of SEQ ID NO: 19; HC-CDR3 having the amino acid sequence of SEQ ID NO: 20 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (3) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33; HC-CDR2 having the amino acid sequence of SEQ ID NO: 34; HC-CDR3 having the amino acid sequence of SEQ ID NO: 35 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (4) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 49 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (5) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (6) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 83 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (6) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 83 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (7) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 86 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (8) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 95; HC-CDR3 having the amino acid sequence of SEQ ID NO: 96 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (9) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108; HC-CDR2 having the amino acid sequence of SEQ ID NO: 109; HC-CDR3 having the amino acid sequence of SEQ ID NO: 110 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (10) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 122 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (11) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 132; HC-CDR2 having the amino acid sequence of SEQ ID NO: 133; HC-CDR3 having the amino acid sequence of SEQ ID NO: 134 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (12) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 146; HC-CDR2 having the amino acid sequence of SEQ ID NO: 147; HC-CDR3 having the amino acid sequence of SEQ ID NO: 148 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (13) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 156 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (14) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 166; HC-CDR2 having the amino acid sequence of SEQ ID NO: 167; HC-CDR3 having the amino acid sequence of SEQ ID NO: 168 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (15) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 186; HC-CDR3 having the amino acid sequence of SEQ ID NO: 187 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (16) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 199; HC-CDR3 having the amino acid sequence of SEQ ID NO: 200 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (17) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 211; HC-CDR3 having the amino acid sequence of SEQ ID NO: 212 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (18) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 222; HC-CDR2 having the amino acid sequence of SEQ ID NO: 223; HC-CDR3 having the amino acid sequence of SEQ ID NO: 224 or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid; (19) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 243; HC-CDR3 having the amino acid sequence of SEQ ID NO: 244 or variants thereof in which one, two, or three amino acids in HC-CDR1, and / or one, two, or three amino acids in HC-CDR2, and / or one, two, or three amino acids in HC-CDR3 are replaced by another amino acid. The VH region comprises one of the following:

[0068] In some embodiments, the antigen-binding molecule is selected from the group consisting of (20) to (37) below: (20) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 5; HC-FR2 having the amino acid sequence of SEQ ID NO: 6; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (21) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 21; HC-FR2 having the amino acid sequence of SEQ ID NO: 22; HC-FR3 having the amino acid sequence of SEQ ID NO: 23; HC-FR4 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (22) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 36; HC-FR2 having the amino acid sequence of SEQ ID NO: 37; HC-FR3 having the amino acid sequence of SEQ ID NO: 38; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (23) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 50; HC-FR2 having the amino acid sequence of SEQ ID NO: 6; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 51 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (24) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 64; HC-FR2 having the amino acid sequence of SEQ ID NO: 65; HC-FR3 having the amino acid sequence of SEQ ID NO: 66; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (25) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 84; HC-FR2 having the amino acid sequence of SEQ ID NO: 6; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (26) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 87; HC-FR2 having the amino acid sequence of SEQ ID NO: 6; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 51 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (27) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 97; HC-FR2 having the amino acid sequence of SEQ ID NO: 98; HC-FR3 having the amino acid sequence of SEQ ID NO: 66; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (28) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 111; HC-FR2 having the amino acid sequence of SEQ ID NO: 112; HC-FR3 having the amino acid sequence of SEQ ID NO: 113; HC-FR4 having the amino acid sequence of SEQ ID NO: 51 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (29) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 123; HC-FR2 having the amino acid sequence of SEQ ID NO: 6; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 51 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (30) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 135; HC-FR2 having the amino acid sequence of SEQ ID NO: 136; HC-FR3 having the amino acid sequence of SEQ ID NO: 137; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (31) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 149; HC-FR2 having the amino acid sequence of SEQ ID NO: 150; HC-FR3 having the amino acid sequence of SEQ ID NO: 151; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (32) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 87; HC-FR2 having the amino acid sequence of SEQ ID NO: 169; HC-FR3 having the amino acid sequence of SEQ ID NO: 168; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (33) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 188; HC-FR2 having the amino acid sequence of SEQ ID NO: 189; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (34) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 201; HC-FR2 having the amino acid sequence of SEQ ID NO: 202; HC-FR3 having the amino acid sequence of SEQ ID NO: 203; HC-FR4 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (35) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 213; HC-FR2 having the amino acid sequence of SEQ ID NO: 214; HC-FR3 having the amino acid sequence of SEQ ID NO: 7; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (36) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 225; HC-FR2 having the amino acid sequence of SEQ ID NO: 226; HC-FR3 having the amino acid sequence of SEQ ID NO: 227; HC-FR4 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid; (37) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 245; HC-FR2 having the amino acid sequence of SEQ ID NO: 246; HC-FR3 having the amino acid sequence of SEQ ID NO: 190; HC-FR4 having the amino acid sequence of SEQ ID NO: 39 or variants thereof in which one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid. The VH region comprises one of the following:

[0069] In some embodiments, the antigen-binding molecule comprises a VH region comprising a CDR according to any one of (1) to (19) above and a FR according to any one of (20) to (37) above.

[0070] In some embodiments, the antigen-binding molecule is selected from the group consisting of (38) to (56) below: (38) A VH region comprising the CDRs according to (1) and the FRs according to (20); (39) A VH region comprising CDRs according to (2) and FRs according to (21); (40) A VH region comprising CDRs according to (3) and FRs according to (22); (41) A VH region comprising CDRs according to (4) and FRs according to (23); (42) a VH region comprising CDRs according to (5) and FRs according to (24); (43) a VH region comprising CDRs according to (6) and FRs according to (25); (44) A VH region comprising CDRs according to (7) and FRs according to (26); (45) A VH region comprising CDRs according to (8) and FRs according to (27); (46) A VH region comprising CDRs according to (9) and FRs according to (28); (47) A VH region comprising CDRs according to (10) and FRs according to (29); (48) A VH region comprising CDRs according to (11) and FRs according to (30); (49) a VH region comprising CDRs according to (12) and FRs according to (31); (50) a VH region comprising CDRs according to (13) and FRs according to (26); (51) A VH region comprising CDRs according to (14) and FRs according to (32); (52) a VH region comprising CDRs according to (15) and FRs according to (33); (53) a VH region comprising CDRs according to (16) and FRs according to (34); (54) A VH region comprising CDRs according to (17) and FRs according to (35); (55) A VH region comprising CDRs according to (18) and FRs according to (36); (56) A VH region comprising CDRs from (19) and FRs from (37) The VH region comprises one of the following:

[0071] In some embodiments, the antigen-binding molecule is selected from the group consisting of (57) to (75) below: (57) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1; (58) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17; (59) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32; (60) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47; (61) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 60; (62) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82; (63) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; (64) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94; (65) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 107; (66) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 121; (67) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 131; (68) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 145; (69) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 155; (70) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 165; (71) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 184; (72) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 198; (73) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 210; (74) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 221; (75) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 242. The VH region comprises one of the following:

[0072] In some embodiments, the antigen-binding molecule is selected from the group consisting of (76) to (97) below: (76) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 10; LC-CDR2 having the amino acid sequence of SEQ ID NO: 11; LC-CDR3 having the amino acid sequence of SEQ ID NO: 12 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (77) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 25; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 27 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (78) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 43 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (79) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 53; LC-CDR2 having the amino acid sequence of SEQ ID NO: 54; LC-CDR3 having the amino acid sequence of SEQ ID NO: 55 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (80) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 68; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 69 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (81) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 74 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (82) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 78; LC-CDR2 having the amino acid sequence of SEQ ID NO: 79; LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (83) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 89; LC-CDR2 having the amino acid sequence of SEQ ID NO: 11; LC-CDR3 having the amino acid sequence of SEQ ID NO: 90 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (84) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 101; LC-CDR2 having the amino acid sequence of SEQ ID NO: 102; LC-CDR3 having the amino acid sequence of SEQ ID NO: 103 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (85) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 115; LC-CDR2 having the amino acid sequence of SEQ ID NO: 116; LC-CDR3 having the amino acid sequence of SEQ ID NO: 117 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (86) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 125; LC-CDR2 having the amino acid sequence of SEQ ID NO: 126; LC-CDR3 having the amino acid sequence of SEQ ID NO: 127 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (87) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 139; LC-CDR2 having the amino acid sequence of SEQ ID NO: 140; LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (88) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 153 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (89) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 158; LC-CDR2 having the amino acid sequence of SEQ ID NO: 159; LC-CDR3 having the amino acid sequence of SEQ ID NO: 160 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (90) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 171; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (91) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 179; LC-CDR2 having the amino acid sequence of SEQ ID NO: 180; LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (92) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73; LC-CDR2 having the amino acid sequence of SEQ ID NO: 26; LC-CDR3 having the amino acid sequence of SEQ ID NO: 194 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (93) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 205; LC-CDR2 having the amino acid sequence of SEQ ID NO: 42; LC-CDR3 having the amino acid sequence of SEQ ID NO: 206 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (94) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 216; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 217 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (95) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229; LC-CDR2 having the amino acid sequence of SEQ ID NO: 172; LC-CDR3 having the amino acid sequence of SEQ ID NO: 230 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (96) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235; LC-CDR2 having the amino acid sequence of SEQ ID NO: 236; LC-CDR3 having the amino acid sequence of SEQ ID NO: 237 or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid; (97) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 248; LC-CDR2 having the amino acid sequence of SEQ ID NO: 249; LC-CDR3 having the amino acid sequence of SEQ ID NO: 250 or variants thereof in which one, two, or three amino acids in LC-CDR1, and / or one, two, or three amino acids in LC-CDR2, and / or one, two, or three amino acids in LC-CDR3 are replaced by another amino acid. The VL region comprises one of:

[0073] In some embodiments, the antigen-binding molecule is selected from the group consisting of (98) to (119) below: (98) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 13; LC-FR2 having the amino acid sequence of SEQ ID NO: 14; LC-FR3 having the amino acid sequence of SEQ ID NO: 15 LC-FR4 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (99) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 28; LC-FR2 having the amino acid sequence of SEQ ID NO: 29; LC-FR3 having the amino acid sequence of SEQ ID NO: 30 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (100) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 44; LC-FR2 having the amino acid sequence of SEQ ID NO: 45; LC-FR3 having the amino acid sequence of SEQ ID NO: 46 LC-FR4 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (101) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 56; LC-FR2 having the amino acid sequence of SEQ ID NO: 57; LC-FR3 having the amino acid sequence of SEQ ID NO: 58 LC-FR4 having the amino acid sequence of SEQ ID NO: 59 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (102) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 28; LC-FR2 having the amino acid sequence of SEQ ID NO: 70; LC-FR3 having the amino acid sequence of SEQ ID NO: 71 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (103) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 28; LC-FR2 having the amino acid sequence of SEQ ID NO: 75; LC-FR3 having the amino acid sequence of SEQ ID NO: 76 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (104) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 28; LC-FR2 having the amino acid sequence of SEQ ID NO: 81; LC-FR3 having the amino acid sequence of SEQ ID NO: 76 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (105) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 91; LC-FR2 having the amino acid sequence of SEQ ID NO: 92; LC-FR3 having the amino acid sequence of SEQ ID NO: 93 LC-FR4 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (106) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 104; LC-FR2 having the amino acid sequence of SEQ ID NO: 105; LC-FR3 having the amino acid sequence of SEQ ID NO: 106 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (107) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 118; LC-FR2 having the amino acid sequence of SEQ ID NO: 119; LC-FR3 having the amino acid sequence of SEQ ID NO: 120 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (108) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 128; LC-FR2 having the amino acid sequence of SEQ ID NO: 129; LC-FR3 having the amino acid sequence of SEQ ID NO: 130 LC-FR4 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (109) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 141; LC-FR2 having the amino acid sequence of SEQ ID NO: 142; LC-FR3 having the amino acid sequence of SEQ ID NO: 143 LC-FR4 having the amino acid sequence of SEQ ID NO: 144 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (110) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 44; LC-FR2 having the amino acid sequence of SEQ ID NO: 45; LC-FR3 having the amino acid sequence of SEQ ID NO: 46 LC-FR4 having the amino acid sequence of SEQ ID NO: 154 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (111) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 161; LC-FR2 having the amino acid sequence of SEQ ID NO: 162; LC-FR3 having the amino acid sequence of SEQ ID NO: 163 LC-FR4 having the amino acid sequence of SEQ ID NO: 164 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (112) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 174; LC-FR2 having the amino acid sequence of SEQ ID NO: 175; LC-FR3 having the amino acid sequence of SEQ ID NO: 176 LC-FR4 having the amino acid sequence of SEQ ID NO: 177 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (113) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 181; LC-FR2 having the amino acid sequence of SEQ ID NO: 182; LC-FR3 having the amino acid sequence of SEQ ID NO: 183 LC-FR4 having the amino acid sequence of SEQ ID NO: 177 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (114) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 28; LC-FR2 having the amino acid sequence of SEQ ID NO: 75; LC-FR3 having the amino acid sequence of SEQ ID NO: 197 LC-FR4 having the amino acid sequence of SEQ ID NO: 177 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (115) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 44; LC-FR2 having the amino acid sequence of SEQ ID NO: 207; LC-FR3 having the amino acid sequence of SEQ ID NO: 208 LC-FR4 having the amino acid sequence of SEQ ID NO: 209 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (116) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 218; LC-FR2 having the amino acid sequence of SEQ ID NO: 219; LC-FR3 having the amino acid sequence of SEQ ID NO: 220 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (117) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 231; LC-FR2 having the amino acid sequence of SEQ ID NO: 232; LC-FR3 having the amino acid sequence of SEQ ID NO: 233 LC-FR4 having the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (118) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 238; LC-FR2 having the amino acid sequence of SEQ ID NO: 239; LC-FR3 having the amino acid sequence of SEQ ID NO: 240 LC-FR4 having the amino acid sequence of SEQ ID NO: 241 or a variant thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid; (119) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 231; LC-FR2 having the amino acid sequence of SEQ ID NO: 251; LC-FR3 having the amino acid sequence of SEQ ID NO: 252 LC-FR4 having the amino acid sequence of SEQ ID NO: 253 or variants thereof in which one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid. The VL region comprises one of:

[0074] In some embodiments, the antigen-binding molecule comprises a VL region comprising a CDR according to any one of (76) to (97) above and a FR according to any one of (98) to (119) above.

[0075] In some embodiments, the antigen-binding molecule is selected from the group consisting of (120) to (142) below: (120) A VL region comprising CDRs according to (76) and FRs according to (98), (121) A VL region comprising CDRs according to (77) and FRs according to (99), (123) A VL region comprising CDRs according to (78) and FRs according to (100); (124) A VL region comprising CDRs according to (79) and FRs according to (101), (125) A VL region comprising CDRs according to (80) and FRs according to (102), (126) A VL region comprising CDRs according to (81) and FRs according to (103), (127) A VL region comprising CDRs according to (82) and FRs according to (104), (128) A VL region comprising CDRs according to (83) and FRs according to (105), (129) A VL region comprising CDRs according to (84) and FRs according to (106), (130) A VL region comprising CDRs according to (85) and FRs according to (107), (131) A VL region comprising CDRs according to (86) and FRs according to (108), (132) A VL region comprising CDRs according to (87) and FRs according to (109), (133) A VL region comprising CDRs according to (88) and FRs according to (110), (134) A VL region comprising CDRs according to (89) and FRs according to (111), (135) A VL region comprising CDRs according to (90) and FRs according to (112), (136) A VL region comprising CDRs according to (91) and FRs according to (113), (137) A VL region comprising CDRs according to (92) and FRs according to (114), (138) A VL region comprising CDRs according to (93) and FRs according to (115), (139) A VL region comprising CDRs according to (94) and FRs according to (116), (140) A VL region comprising CDRs according to (95) and FRs according to (117), (141) A VL region comprising CDRs according to (96) and FRs according to (118), (142) VL region containing CDRs from (97) and FRs from (119) The VL region comprises one of:

[0076] In some embodiments, the antigen-binding molecule is selected from the group consisting of (143) to (164) below: (143) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9; (144) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24; (145) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40; (146) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 52; (147) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67; (148) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72; (149) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77; (150) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88; (151) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100; (152) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 114; (153) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 124; (154) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 138; (155) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152; (156) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 157; (157) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 170; (158) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 178; (159) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 191; (160) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 204; (161) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 215; (162) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 228; (163) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 234; (164) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 247. The VL region comprises one of:

[0077] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1) to (75) above, and a VL region according to any one of (76) to (164) above. In embodiments according to the present disclosure, one or more amino acids are substituted with another amino acid. Substitutions include replacing an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue for a substitution according to the present disclosure may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, and is selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid may be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those recited in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. 202 (1991) pp. 301-336.

[0078] In some embodiments, substitutions may be biochemically conservative. In some embodiments, where the substituted amino acid is provided in one of lines 1-5 of the table below, the replacement amino acid for the substitution is another non-identical amino acid provided in the same line.

[0079] [Table 1]

[0080] Illustratively, in some embodiments where the substitution is for a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0081] In some embodiments, the replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces, hi some embodiments, the replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces.

[0082] [Table 2]

[0083] That is, in some aspects, a non-polar amino acid is substituted with another non-identical non-polar amino acid. In some aspects, a polar amino acid is substituted with another non-identical polar amino acid. In some aspects, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid. In some aspects, a basic polar amino acid is substituted with another non-identical basic polar amino acid. In some aspects, a neutral amino acid is substituted with another non-identical neutral amino acid. In some aspects, a positively charged amino acid is substituted with another non-identical positively charged amino acid. In some aspects, a negatively charged amino acid is substituted with another non-identical negatively charged amino acid.

[0084] In some embodiments, substitutions may be functionally conservative, i.e., the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule comprising the substitution, compared to an equivalent unsubstituted molecule.

[0085] The VH and VL regions of the antigen-binding region of an antibody together constitute an Fv region. In some embodiments, an antigen-binding molecule according to the present disclosure comprises or consists of an Fv region that binds to CNX. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), linked by a linker region.

[0086] The VL and light chain constant (CL) regions, and the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of an antibody together constitute a Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CH1 (e.g., a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CL (e.g., a VH-CL fusion polypeptide), and a polypeptide comprising a VL and CH (e.g., a VL-CH1 fusion polypeptide). That is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of a Fab or CrossFab are provided as a single polypeptide linked by a linker region, i.e., a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0087] In some embodiments, the antigen-binding molecules described herein comprise or consist of a whole antibody that binds to CNX. As used herein, "whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Various types of immunoglobulins and their structures are described in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is incorporated herein by reference in its entirety.

[0088] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that contain two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3), and similarly, the light chain contains a VL followed by a CL. Depending on the heavy chain, immunoglobulins are classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0089] In some embodiments, the antigen-binding molecules described herein comprise or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to CNX.

[0090] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more regions (e.g., CH1, CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0091] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 254, 259, 260, or 263.

[0092] In some embodiments, the antigen-binding molecule comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 255 or 261. In some embodiments, the antigen-binding molecule comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 257. In some embodiments, the antigen-binding molecule comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 258 or 262.

[0093] In some embodiments, the antigen-binding molecule comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 256, 264, 265, or 266.

[0094] It will be appreciated that the CH2 and / or CH3 regions may contain further substitutions in accordance with the modifications of the Fc region of the antigen binding molecules described herein. In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0095] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 267, 268, 269, 270, 271, or 272.

[0096] In some aspects, the antigen-binding molecule is or comprises a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the antigen-binding molecule is or comprises a fully human antibody / antibody fragment. A fully human antibody / antibody fragment may be encoded by a human nucleic acid sequence. A fully human antibody / antibody fragment may lack non-human amino acid sequences. Commonly adopted approaches for producing fully human antibodies include (i) phage display, in which human antibody genes are expressed in a phage display library, and (ii) production of antibodies in transgenic mice engineered to harbor human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56:369-421). Briefly, in the human antibody gene phage display approach, genes encoding VH and VL chains are generated from "naive" human lymphocytes by PCR amplification and cloning, assembled into libraries, and expressed as disulfide-linked Fab fragments or single-chain Fv (scFv) fragments. Genes encoding Fab or scFv are fused to surface coat proteins of filamentous bacteriophage, and Fab or scFv capable of binding to the desired target can be identified by screening libraries with antigen. To enhance the affinity of the Fab / scFv fragments, molecular evolution or affinity maturation procedures can be employed. In the transgenic mouse approach, mice in which endogenous mouse Ig loci have been replaced with their human homologs by homologous recombination are immunized with antigen, and monoclonal antibodies are prepared using conventional hybridoma technology to obtain fully human monoclonal antibodies.

[0097] In some embodiments, the antigen-binding molecules of the present disclosure are mouse antibodies / antibody fragments. In some embodiments, the antibodies / antibody fragments are obtained by phage display methods using a human naive antibody gene library.

[0098] In some embodiments, the antigen-binding molecule is a mouse / human chimeric antibody / antibody fragment (i.e., an antigen-binding molecule comprising the variable domains of a mouse antibody and the constant region of a human antibody). In some embodiments, the antigen-binding molecule is a humanized antibody / antibody fragment. In some embodiments, the antigen-binding molecule comprises the CDRs of a mouse antibody and the framework and constant regions of a human antibody.

[0099] Mouse / human chimeric antigen-binding molecules can be prepared from mouse antibodies by a chimerization process, for example, as described in Chapter 8, particularly Chapter 8, Section 3, of Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (ed.), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014).

[0100] Humanized antigen-binding molecules can be prepared from mouse antibodies by a humanization process, as described, for example, in Chapter 7, particularly Section 3.1, "Antibody Humanization," of "Human Monoclonal Antibodies: Methods and Protocols," edited by Michael Steinitz, Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014). Antibody humanization techniques are also described, for example, in Safdari et al., Biotechnol Genet Eng Rev (2013) 29:175-86.

[0101] Aspects of the present disclosure relate to multispecific antigen-binding molecules. "Multispecific" means that an antigen-binding molecule exhibits specific binding to two or more targets. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains comprising, for example, non-identical VH and VL).

[0102] In some embodiments, the antigen-binding molecule binds to CNX and another target (e.g., an antigen other than CNX), and is therefore at least bispecific. The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0103] It will be appreciated that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present disclosure may include antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules that bind to CNX and antigens other than CNX may include (i) antigen-binding molecules that bind to CNX and (ii) antigen-binding molecules that bind to antigens other than CNX.

[0104] It will also be recognized that an antigen-binding molecule (e.g., a multispecific antigen-binding molecule) according to the present disclosure may comprise an antigen-binding polypeptide or antigen-binding polypeptide complex capable of binding to a target for which the antigen-binding molecule is specific.

[0105] In some embodiments, an antigen-binding molecule that is a component of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as an "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0106] In some embodiments, the antigen other than CNX in the multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or hormone.

[0107] A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be aberrantly expressed by cancer cells (e.g., the cancer cell antigen can be expressed with an abnormal localization) or with an abnormal structure. A cancer cell antigen can elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the antigen-binding molecules described herein is displayed on the outer surface (i.e., extracellularly) of a cancer cell. A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of a cancer symptom. A cancer-associated antigen can be associated with the cause or etiology of cancer, or can be aberrantly expressed as a consequence of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, e.g., compared to the level of expression by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen is preferentially expressed by cancer cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen can be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen can be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or a cell surface glycolipid or glycoprotein.

[0108] An immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or at the cell surface of an immune cell. In some embodiments, the portion of the immune cell surface molecule bound by an antigen-binding molecule of the present disclosure is present on the outer surface (i.e., extracellular) of the immune cell. An immune cell surface molecule is expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a natural killer (NK) cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor cell thereof (e.g., a thymocyte or a pre-B cell).

[0109] In some embodiments, the antigen-binding molecule is an immune cell engager. Immune cell engagers are reviewed in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418-434, and Ellerman, Methods (2019) 154:102-117, the entire contents of which are incorporated herein by reference. Immune cell engager molecules contain an antigen-binding region for a target antigen of interest and an antigen-binding region for recruitment / engagement of immune cells of interest. Immune cell engagers recruit and engage immune cells through an antigen-binding region specific for an immune cell surface molecule.

[0110] The most well-studied immune cell engagers are bispecific T cell engagers (BiTEs), which contain a target antigen-binding domain and a CD3 polypeptide (typically CD3ε)-binding domain through which BiTEs recruit T cells. Binding of a BiTE to its target antigen and the CD3 polypeptide expressed by T cells results in T cell activation and ultimately directs T cell effector activity toward cells expressing the target antigen. Other types of immune cell engagers are known in the art, including natural killer cell engagers such as bispecific killer engagers (BiKEs), which recruit and activate NK cells.

[0111] In some embodiments, the multispecific antigen-binding molecules described herein exhibit at least monovalent binding for CNX and also exhibit at least monovalent binding for a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε). In some embodiments, the antigen-binding molecule comprises one binding site for CNX and one binding site for a CD3 polypeptide.

[0112] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε). In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε). In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε). That is, in some embodiments, the antigen-binding molecule comprises the VH region and VL region of an antigen-binding molecule that binds to a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε).

[0113] In some embodiments, the antigen-binding molecule comprises the CDR, FR, and / or VH and / or VL regions of a CD3 polypeptide-binding antibody clone, or the CDR, FR, and / or VH and / or VL regions derived therefrom of a CD3 polypeptide-binding antibody clone.

[0114] In some embodiments, the CD3 polypeptide-binding antibody clone is selected from OKT3 (Kjer-Nielsen et al., PNAS (2004) 101(20):7675-80), SP34 (e.g., as described in WO 2014 / 122143 A1), UCHT1 (e.g., as described in WO 2000 / 041474 A1), HIT3a (Invitrogen Cat#16-0039-85), and clone SK7 (Invitrogen Cat#16-0036-81).

[0115] In some embodiments, the immune cells engaged by the immune cell engager are T cells or NK cells, hi some embodiments, the immune cell engager is a T cell engager. Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which is incorporated herein by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, such as IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, such as IgG, chimeric IgG, κλ-body; General HC; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; "variable domain only" bispecific antigen binding molecules, e.g., tandem scFv (taFV), triple body, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAb, triple head, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion proteins, e.g., scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g., scFv- Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEED body), taFv-Fc(ki h), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEED body), DART-Fc, scFv-CH3(kih), TriFab; Fc fusions such as Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions such as Dia-diabody, scDb-CH3; IgE / IgM CH2 fusions such as scFv-EHD2-scFv, scFvMHD2-scFv;Fab fusion proteins, e.g., Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g., DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g., IgG(kih), IgG(kih) general LC, ZW1 IgG general LC, Bichronix general LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEED body, Duobody, four-in-one CrossMab(kih), LUZ-Y general LC; LUZ-Y scFab-IgG, FcFc*; adducted and Fc-modified IgG, e.g., IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-Ig, DVI-Ig (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g., Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEED body, TriFab; adducted IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fa b, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-IgG (CαCβFab), Fab-IgG (CR3), Fab-hinge-IgG (CR3); additional IgG-LC fusions, such as IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; additional IgG-HC and LC fusions, such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, such as Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, such as F(ab')2-scFv2; CH1 / CL fusion proteins, such as scFv2-CH1-hinge / CL;modified IgG, such as DAF (two-in-one IgG), DutaMab, Mab; 2 and non-Ig fusions, such as DNL-Fab4-IgG.

[0116] Those skilled in the art can design and prepare bispecific antigen-binding molecules. Methods for producing multispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments with a reducible disulfide bond or a non-reducible thioether bond, as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding Molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, the entire contents of which are incorporated herein by reference. For example, N-succinimidyl-3-(-2-pyridyldithio)propionate (SPDP) can be used to chemically crosslink, for example, Fab fragments via SH groups in the hinge region to create disulfide-linked bispecific F(ab)2 heterodimers.

[0117] Other methods for producing multispecific antigen-binding molecules include fusing antibody-producing hybridomas with, for example, polyethylene glycol, to produce quadroma cells capable of secreting bispecific antibodies, as described, for example, in DM and Bast, BJ 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

[0118] Multispecific antigen-binding molecules according to the present disclosure can also be produced recombinantly by expression from nucleic acid constructs encoding the polypeptides for the antigen-binding molecule, e.g., as described in Chapter 40, "Production of Bispecific Antigen-binding Molecules: Diabodies and Tandem scFv" (Hornig and Farber-Schwarz) of Antibody Engineering: Methods and Protocols, 2nd Edition, (Humana Press, 2012), the contents of which are incorporated herein by reference in their entirety, or French, "How to make bispecific antigen-binding molecules," Methods Mol. Med. 2000;40:333-339.

[0119] For example, a DNA construct encoding the light and heavy chain variable domains for two antigen-binding fragments (i.e., the light and heavy chain variable domains for the antigen-binding fragment capable of binding to CNX and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein), as well as a sequence encoding a suitable linker or dimerization domain between the antigen-binding fragments, can be prepared by molecular cloning techniques. The construct can then be expressed (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell) to produce a recombinant bispecific antibody, which can optionally be purified. Fc area In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region.

[0120] The Fc region consists of a CH2 and CH3 region from one polypeptide and a CH2 and CH3 region from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.

[0121] Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), membrane attack complex (MAC) formation, cellular degranulation, cytokine and / or chemokine production, and antigen processing and presentation. Modifications to antibody Fc regions that affect Fc-mediated functions are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, incorporated herein by reference in its entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region comprising a modification that increases or decreases an Fc-mediated function compared to an antigen-binding molecule comprising a corresponding unmodified Fc region.

[0122] When an Fc region / CH2 / CH3 is described as containing a modification "corresponding to" a reference substitution, the equivalent substitution in the homologous Fc / CH2 / CH3 is intended. By way of illustration, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) corresponds to the L to A substitution at positions 117 and 118 of the mouse Ig gamma-2 A chain C region (UniProtKB: P01863-1, v1).

[0123] Where an Fc region is described as including a modification, the modification can be present in one or both of the polypeptide chains that together form the Fc region. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that comprises a modification. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that comprises a modification in one or more of the CH2 and / or CH3 regions.

[0124] In some embodiments, the Fc region comprises a modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises a modification to increase ADCC. In some embodiments, the Fc region comprises a modification to increase ADCP. In some embodiments, the Fc region comprises a modification to increase CDC. Antigen-binding molecules comprising an Fc region comprising a modification to increase an Fc-mediated function (e.g., ADCC, ADCP, CDC) elicit an increased level of the associated effector function compared to a corresponding antigen-binding molecule comprising an unmodified Fc region.

[0125] In some embodiments, the Fc region comprises a modification to increase binding to an Fc receptor. In some embodiments, the Fc region comprises a modification to increase binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIa. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIb. In some embodiments, the Fc region comprises a modification to increase binding to FcRn. In some embodiments, the Fc region comprises a modification to increase binding to complement proteins. In some embodiments, the Fc region comprises a modification to increase binding to C1q. In some embodiments, the Fc region comprises modifications to promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises modifications to increase the half-life of the antigen-binding molecule. In some embodiments, the Fc region comprises modifications to increase co-association.

[0126] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions F243L / R292P / Y300L / V305I / P396L, as described in Stavenhagen et al., Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S239D / I332E or S239D / I332E / A330L, as described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S298A / E333A / K334A, as described in Shields et al., J Biol Chem. (2001) 276:6591-6604. In some embodiments, the Fc region comprises modifications in one of the heavy chain polypeptides corresponding to the combination of substitutions L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and modifications in the other heavy chain polypeptide corresponding to the combination of substitutions D270E / K326D / A330M / K334E, as described in Mimoto et al., MAbs. (2013):5:229-236. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527.

[0127] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions K326W / E333S, as described in Idusogie et al. J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S267E / H268F / S324T, as described in Moore et al. MAbs. (2010) 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions described in Natsume et al. Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions E345R / E430G / S440Y, as described in Diebolder et al. Science (2014) 343(6176):1260-3.

[0128] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions M252Y / S254T / T256E as described in Dall'Acqua et al. J Immunol. (2002) 169:5171-5180. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions M428L / N434S as described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159.

[0129] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S267E / L328F as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions N325S / L328F as described in Shang et al., Biol Chem. (2014) 289:15309-15318.

[0130] In some embodiments, the Fc region comprises a modification to reduce / prevent an Fc-mediated function. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCC. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCP. In some embodiments, the Fc region comprises a modification to reduce / prevent CDC. Antigen-binding molecules comprising an Fc region comprising a modification to reduce / prevent an Fc-mediated function (e.g., ADCC, ADCP, CDC) elicit a reduced level of the associated effector function compared to a corresponding antigen-binding molecule comprising an unmodified Fc region.

[0131] In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fc receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to complement proteins. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to C1q. In some aspects, the Fc region comprises a modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0132] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to induce a relevant Fc-mediated function). Accordingly, an antigen-binding molecule comprising such an Fc region also lacks the ability to induce the relevant function. Such an antigen-binding molecule may be described as lacking the relevant function.

[0133] In some embodiments, the Fc region is incapable of inducing ADCC. In some embodiments, the Fc region is incapable of inducing ADCP. In some embodiments, the Fc region is incapable of inducing CDC. In some embodiments, the Fc region is incapable of inducing ADCC and / or incapable of inducing ADCP and / or incapable of inducing CDC.

[0134] In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcγ receptor. In some embodiments, the Fc region is unable to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is unable to bind to FcγRIIIa. In some embodiments, the Fc region is unable to bind to FcγRIIa. In some embodiments, the Fc region is unable to bind to FcγRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to complement proteins. In some embodiments, the Fc region is unable to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0135] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G, as described in Leabman et al., MAbs. (2013) 5:896-903. In some embodiments, the Fc region comprises a modification corresponding to L235E, as described in Alegre et al., J Immunol. (1992) 148:3461-3468. In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A or F234A / L235A, as described in Xu et al., Cell Immunol. (2000) 200:16-26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G, as described in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S228P / L235E, as described in Newman et al., Clin. Immunol. (2001) 98:164-174. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions H268Q / V309L / A330S / P331S, as described in An et al., MAbs. (2009) 1:572-579. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions V234A / G237A / P238S / H268A / V309L / A330S / P331S, as described in Vafa et al., Methods. (2014) 65:114-126. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331S, as described in US 2015 / 0044231 A1.

[0136] The combination of substitutions "L234A / L235A" and corresponding substitutions (e.g., F234A / L235A in human IgG4) is known to disrupt Fc binding to Fcγ receptors, inhibit ADCC, ADCP, and reduce C1q binding and therefore CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, the entire contents of which are incorporated herein by reference). The substitutions "P329G" and "P329A" reduce C1q binding (and thereby CDC). Substitution of "N297" with "A," "G," or "Q" is known to eliminate glycosylation, thereby reducing Fc binding to C1q and Fcγ receptors, and therefore CDC and ADCC. Lo et al. J. Biol. Chem (2017) 292(9):3900-3908 (incorporated herein by reference in its entirety) report that the combination of substitutions L234A / L235A / P329G abrogates complement binding and fixation, as well as Fcγ receptor-dependent, antibody-dependent cell-mediated cytotoxicity, in both mouse IgG2a and human IgG1.

[0137] It is disclosed in US 2015 / 0044231 A1 that the combination of substitutions L234A / L235E / G237A / A330S / P331S in IgG1 Fc abolishes the induction of phagocytosis, ADCC, and CDC.

[0138] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P, as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The substitution S228P in an IgG4 Fc reduces Fab-arm exchange (which may be undesirable).

[0139] In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to substitution P329G. In some embodiments, the Fc region comprises a modification corresponding to substitution N297Q.

[0140] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G / N297Q.

[0141] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331S. In some embodiments, the Fc region comprises a modification corresponding to, for example, the substitution S228P in IgG4.

[0142] In some embodiments, particularly in embodiments where the antigen-binding molecule is a multispecific (e.g., bispecific) antigen-binding molecule, the antigen-binding molecule comprises an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote assembly of the Fc regions. Recombinant coexpression and subsequent assembly of the component polypeptides of an antigen-binding molecule results in several possible combinations. To improve the yield of a desired combination of polypeptides in a recombinant production of an antigen-binding molecule, it is advantageous to introduce modifications to the Fc region that promote assembly of a desired combination of heavy chain polypeptides. The modifications may, for example, promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immunol (2016) 7:394, the entire contents of which are incorporated herein by reference.

[0143] In some embodiments, an antigen-binding molecule of the disclosure comprises an Fc region comprising a paired substitution in the CH3 region of the Fc region with one of the following formats: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED, or A107, as shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394. Polypeptides and Particularly Exemplary Antigen-Binding Molecules The present disclosure also provides polypeptide components of the antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.

[0144] The antigen-binding molecules of the present disclosure may be complexes of or comprise polypeptides. Where a polypeptide comprises more than one domain or region, it will be appreciated that the multiple domains / regions may preferably be present in the same polypeptide chain, i.e., a polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains / regions.

[0145] In some aspects, a polypeptide according to the present disclosure comprises or consists of a VH described herein. In some aspects, a polypeptide according to the present disclosure comprises or consists of a VL described herein.

[0146] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide further comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises an immunoglobulin (Ig) CH1, CH2, and / or CH3 region.

[0147] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein.

[0148] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence, hi some embodiments, the polypeptide comprises a CL region as described herein. In some aspects, a polypeptide according to the present disclosure comprises: (i) VH (ii) VL (iii) VH-CH1 (iv) VL-CL (v) VL-CH1 (vi) VH-CL (vii) VH-CH1-CH2-CH3 (viii) VL-CL-CH2-CH3 (ix) VL-CH1-CH2-CH3 (x)VH-CL-CH2-CH3 The structure from N-terminus to C-terminus is one of:

[0149] Also provided by the present disclosure are antigen-binding molecules consisting of the polypeptides of the present disclosure. The antigen-binding molecules of the present disclosure comprise one of the following polypeptide combinations:

[0150] (A) VH+VL (B) VH-CH1+VL-CL (C) VL-CH1+VH-CL (D) VH-CH1-CH2-CH3+VL-CL (E) VH-CL-CH2-CH3+VL-CH1 (F) VL-CH1-CH2-CH3+VH-CL (G)VL-CL-CH2-CH3+VH-CH1 (H)VH-CH1-CH2-CH3+VL-CL-CH2-CH3 (I)VH-CL-CH2-CH3+VL-CH1-CH2-CH3 In some embodiments, the antigen-binding molecule comprises a combination of two or more polypeptides shown in (A) to (I) above. For example, with respect to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3 and two polypeptides comprising the structure VL-CL.

[0151] In some embodiments, the antigen-binding molecules of the present disclosure comprise one of the following polypeptide combinations: (J)VH (anti-CNX) + VL (anti-CNX) (K)VH(anti-CNX)-CH1+VL(anti-CNX)-CL (L)VL(anti-CNX)-CH1+VH(anti-CNX)-CL (M)VH(anti-CNX)-CH1-CH2-CH3+VL(anti-CNX)-CL (N)VH(anti-CNX)-CL-CH2-CH3+VL(anti-CNX)-CH1 (O)VL(anti-CNX)-CH1-CH2-CH3+VH(anti-CNX)-CL (P)VL(anti-CNX)-CL-CH2-CH3+VH(anti-CNX)-CH1 (Q)VH(Anti-CNX)-CH1-CH2-CH3+VL(Anti-CNX)-CL-CH2-CH3 Here, "VH(anti-CNX)" refers to the VH of an antigen-binding molecule capable of binding to CNX as defined herein, for example, in one of (1) to (75), and "VL(anti-CNX)" refers to the VL of an antigen-binding molecule capable of binding to CNX as defined herein, for example, in one of (76) to (164).

[0152] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1, 17, 32, 47, 60, 82, 85, 94, 107, 121, 131, 145, 155, 165, 184, 198, 210, 221, or 242.

[0153] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 9, 24, 40, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 178, 191, 204, 215, 228, 234, or 247.

[0154] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 273, 276, 279, 282, 285, 290, 292, 295, 298, 301, 304, 307, 310, 313, 317, 320, 323, 326, 330, 274, 277, 280, 283, 286, 291, 293, 296, 299, 302, 305, 308, 311, 314, 318, 321, 324, 327, or 331.

[0155] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 275, 278, 281, 284, 287, 288, 289, 294, 297, 300, 303, 306, 309, 312, 315, 316, 319, 322, 325, 328, 329, or 332.

[0156] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising a VH region comprising the heavy chain CDRs and a VL region of the light chain CDRs of a clone selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, COOl, C008, CO10, C023, C025, C040, C046, and C117, as set forth in Table A herein. That is, in some embodiments, an antigen-binding molecule comprises a polypeptide comprising (i) a VH region comprising the HC-CDRl, HC-CDR2, and HC-CDR3 set forth in column A of Table A, and (ii) a VL region comprising the LC-CDRl, LC-CDR2, and LC-CDR3 set forth in column B of Table A, wherein the sequences in columns A and B are selected from the same row of Table A.

[0157] In some embodiments, antigen-binding molecules of the present disclosure comprise a polypeptide comprising a VH region comprising heavy chain FRs and a VL region comprising light chain FRs of a clone selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, COOl, COO8, CO10, CO23, CO25, CO40, C046, and C117, as set forth in Table B herein. That is, in some embodiments, antigen-binding molecules comprise a polypeptide comprising (i) a VH region comprising HC-FRl, HC-FR2, HC-FR3, and HC-FR4 set forth in column A of Table B, and (ii) a VL region comprising LC-FRl, LC-FR2, LC-FR3, and LC-FR4 set forth in column B of Table B, wherein the sequences in columns A and B are selected from the same row of Table B.

[0158] In some embodiments, antigen-binding molecules of the disclosure comprise a polypeptide comprising: (i) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in column A of Table C; and (ii) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0159] In some embodiments, an antigen-binding molecule of the disclosure comprises a polypeptide comprising the VH and VL regions of a clone selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, COOl, COO8, CO10, CO23, CO25, CO40, C046, and C117, as set forth in Table C herein. That is, in some embodiments, an antigen-binding molecule comprises a polypeptide comprising (i) the amino acid sequence set forth in column A of Table C and (ii) the amino acid sequence set forth in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0160] In some embodiments, antigen-binding molecules of the disclosure include (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in column A of Table D, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in column B of Table D, wherein the sequences in columns A and B are selected from the same row of Table D.

[0161] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide of an antigen-binding molecule according to any one of [1] to

[46] detailed in Table D herein. That is, in some embodiments, the antigen-binding molecule comprises (i) a polypeptide comprising or consisting of the amino acid sequence shown in column A of Table D, and (ii) a polypeptide comprising or consisting of the amino acid sequence shown in column B of Table D, wherein the sequences in columns A and B are selected from the same row of Table D. Linkers and further sequences In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure comprise one or more linker sequences between amino acid sequences, which may be provided at one or both ends of one or more of the VH, VL, CH1-CH2 hinge region, CH2 region, and CH3 region of the antigen-binding molecule / polypeptide.

[0162] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0163] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n (SEQ ID NOs: 410 and 411) or (GxS)nGm (SEQ ID NOs: 412 and 413), where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5, or 6, and m = 0, 1, 2, or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies (e.g., in tandem) of the sequence motif G4S (SEQ ID NO: 414). In some embodiments, the linker sequence comprises or consists of (G4S)4 (SEQ ID NO: 415) or (G4S)6 (SEQ ID NO: 416). In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0164] The antigen-binding molecules and polypeptides of the present disclosure may further comprise additional amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides may comprise an amino acid sequence to facilitate expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecules and polypeptides of the present disclosure may further comprise an amino acid sequence that forms a detectable moiety, for example, as described below.

[0165] The antigen-binding molecules and polypeptides of the present disclosure may further comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain signal peptides.

[0166] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide or may be present in a newly synthesized antigen-binding molecule / polypeptide. The signal peptide provides for efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is often removed by cleavage and therefore is not included in the mature antigen-binding molecule / polypeptide secreted from cells expressing the antigen-binding molecule / polypeptide.

[0167] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176). Labels and conjugates In some aspects, the antigen-binding molecules of the present disclosure further comprise a detectable moiety.

[0168] In some embodiments, the antigen-binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical, a nucleic acid, or an enzymatic label. The antigen-binding molecule may be covalently or non-covalently labeled with the detectable moiety.

[0169] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), chelates of rare earth elements such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radiolabels include hydrogen 3 ,sulfur 35 ,carbon 14 , Phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m ,thulium 165 ,thulium 167 ,thulium168 ,copper 67 , fluorine 18 ,yttrium 90 ,palladium 100 , bismuth 217 , and antimony 211 and radioisotopes such as . Luminescent labels include radioluminescent, chemiluminescent (e.g., acridinium ester, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers.

[0170] In some embodiments, the antigen-binding molecule / polypeptide optionally comprises an epitope tag at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide, such as His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g., biotin, digoxigenin, dinitrophenol).

[0171] In some embodiments, the antigen-binding molecule / polypeptide comprises a moiety having a detectable activity, such as an enzymatic moiety, including, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucorinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0172] In some embodiments, the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect, i.e., a drug moiety. The drug moiety may be a small molecule (e.g., an organic compound with a low molecular weight (less than 1000 daltons, typically about 300-700 daltons)). Drug moieties are described, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety may be or include a cytotoxic agent. In some embodiments, the drug moiety may be or include a chemotherapeutic agent. Drug moieties include, for example, calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[0173] Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, e.g., molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules include an antigen-binding region / domain that comprises or consists of the antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g., provided as an scFv) or Fab region of an antibody, or a whole antibody. For example, antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) that comprise a (cytotoxic) drug moiety. Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules, such as immune cell engager molecules that contain domains for recruiting (effector) immune cells, including BiTE, BiKE, and TriKE (reviewed, e.g., in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418-434, and Ellerman, Methods (2019) 154:102-117, both of which are incorporated herein by reference in their entirety). Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors that provide both antigen-binding and T-cell activation functions (the structure, function, and engineering of CARs are reviewed, e.g., in Dotti et al., Immunol Rev (2014) 257(1), which is incorporated herein by reference in its entirety).

[0174] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a drug moiety. The antigen-binding molecule is conjugated to a drug moiety. Antibody-drug conjugates are generally described, for example, in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14 (incorporated herein by reference in its entirety). FDA-approved ADCs currently on the market are described in Tong et al., Molecules. 2021 Oct; 26(19): 5847 (incorporated herein by reference in its entirety).

[0175] In some embodiments, the antibody-drug conjugate comprises an antigen-binding molecule portion, a drug portion (or payload portion), and a linker that attaches the drug portion to the antibody. In some embodiments, the antibody-drug conjugate consists of an antibody portion, a drug portion (or payload portion), and a linker that attaches the drug portion to the antibody.

[0176] An antigen-binding molecule portion can be a molecule that binds to a given target antigen. Antigen-binding portions include antibodies (i.e., immunoglobulins (Ig)) and antigen-binding fragments thereof. As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antigen-binding molecules derived from antibodies, such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH), etc. Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab')2, and F(ab')2 fragments.

[0177] Linkers can be cleavable or non-cleavable. Linkers can be based on chemical motifs such as disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable). The type of linker, cleavable or non-cleavable, confers specific properties to the cytotoxic drug. For example, a non-cleavable linker retains the drug intracellularly. As a result, the entire antibody, linker, and cytotoxic (anti-cancer) drug enters the target cancer cell, where the antibody is degraded into amino acids. The resulting complex (amino acids, linker, and cytotoxic drug) is thought to be the active drug. In contrast, a cleavable linker is cleaved by enzymes within the cancer cell.

[0178] The drug moiety (or payload) may be a small molecule or nucleic acid drug. In some embodiments, the drug moiety (or payload) is or comprises a cytotoxic agent. In some embodiments, the drug moiety is or comprises a chemotherapeutic agent. In some embodiments, the drug moiety is or comprises an anti-arthritic drug. In some embodiments, the drug moiety is or comprises a steroid. Functional properties of antigen-binding molecules The antigen-binding molecules described herein are characterized by reference to certain functional properties. In some aspects, the antigen-binding molecules described herein exhibit the following properties: binds to CNX (e.g., human CNX and / or mouse CNX); binds to CRT (e.g., human CRT); cross-reactively binds to CNX (e.g., human CNX and / or mouse CNX) and CRT (e.g., human CRT); reducing the function of CNX / CRT and / or complexes containing CNX / CRT; reducing or inhibiting extracellular matrix degradation (e.g., collagen and / or gelatin degradation); reducing or inhibiting the extracellular matrix degrading activity of cells characterized by the expression of CNX; Reducing or inhibiting the extracellular matrix degrading activity of cancer cells; Reduce or inhibit the extracellular matrix degrading activity of fibroblasts; Reduce or inhibit the extracellular matrix degrading activity of synovial fibroblasts; reducing or inhibiting the degradation of extracellular matrix by cells characterized by the expression of CNX; reducing or inhibiting the degradation of extracellular matrix by cancer cells; reducing or inhibiting the degradation of extracellular matrix by fibroblasts; reducing or inhibiting extracellular matrix degradation by synovial fibroblasts; Reduces the activity of oxidoreductase; reducing the activity of disulfide bond reductase; Reduces cartilage breakdown; Increased killing of cells expressing CNX / CRT; Increases ADCC of CNX / CRT-expressing cells; inhibiting tumor growth; reducing or inhibiting cancer metastasis; Increase the survival rate of a subject with cancer; and / or reducing the pathology of a disease / condition characterized by ECM degradation in a subject; reducing the pathology of diseases / conditions characterized by cartilage degradation (e.g., arthritis) in a subject; The compound may possess one or more of:

[0179] It will be recognized that a given antigen-binding molecule may exhibit two or more of the properties recited in the preceding paragraph. A given antigen-binding molecule can be evaluated for the properties recited in the preceding paragraph using a suitable assay. For example, the assay may be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay. In some embodiments, the assay may be, for example, an in vivo assay performed in a non-human animal. In some embodiments, the assay may be, for example, an ex vivo assay performed using cells / tissues / organs obtained from a subject.

[0180] If the assay is a cell-based assay, the assay may include treating cells with a given antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the recited properties. The assay may employ species labeled with a detectable entity to facilitate their detection. The assay may include treating cells individually with a range of amounts / concentrations (e.g., a dilution series) of the given antigen-binding molecule, followed by assessing the recited properties. It will be appreciated that the cells preferably express the target antigen (i.e., CNX / CRT) for the antigen-binding molecule.

[0181] Analysis of the results of such assays may include determining the concentration at which 50% of the maximal level of the relevant activity is reached. The concentration of a given drug at which 50% of the maximal level of the relevant activity is reached may be referred to as the "half maximal effective concentration" of the drug with respect to the relevant activity, which is also known as the "EC 50 Illustratively, the EC of a given antigen-binding molecule for binding to human CNX may be 50 may be the concentration of the antigen-binding molecule at which 50% of the maximal level of binding to human CNX is achieved.

[0182] Depending on the specific 50 is the "half maximal inhibitory concentration" or "IC 50 ", which is the concentration of an agent at which 50% of the maximal level of inhibition of a given property is observed. The antigen-binding molecules described herein bind to CNX. In some embodiments, the antigen-binding molecules bind to CRT. The antigen-binding molecules and antigen-binding domains described herein preferably exhibit specific binding to the relevant target antigen (e.g., CNX). As used herein, "specific binding" refers to binding that is selective for the antigen and can be distinguished from non-specific binding to non-target antigens. Antigen-binding molecules / domains that specifically bind to a target molecule preferably bind to the target with greater affinity and / or longer duration than when binding to other non-target molecules.

[0183] The ability of a given polypeptide to bind to a given molecule can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or radiolabeled antigen binding assay (RIA), enzyme-linked immunosorbent assay analysis. Through such analysis, binding to a given molecule can be measured and quantified. In some embodiments, binding can be a response detected in a given assay.

[0184] In some embodiments, the extent of binding of the antigen-binding molecule to the non-target molecule is less than about 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, the specificity of binding is reflected in terms of binding affinity, and the antigen-binding molecule may be characterized by the dissociation constant (K) of the antigen-binding molecule for the non-target molecule. D ) by at least 0.1 orders of magnitude (i.e., 0.1 × 10 n , where n is an integer representing the order of magnitude) D which may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0185] The binding affinity for a given target antigen for the antigen-binding molecules described herein may be determined by biolayer interferometry, for example, as described in the Examples of the present disclosure.

[0186] In some aspects, the antigen-binding molecules described herein have affinities in the micromolar range, i.e., K D =9.9×10 -4 ~1×10 -6 In some embodiments, the antigen-binding molecules described herein bind to CNX with submicromolar affinity, i.e., K D <1×10 -6 In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the nanomolar range, i.e., K D =9.9×10 -7 ~1×10 -9 In some embodiments, the antigen-binding molecules described herein bind to CNX with sub-nanomolar affinity, i.e., K D <1×10 -9 In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the picomolar range, i.e., K D =9.9×10 -10 ~1×10 -12In some embodiments, the antigen-binding molecules described herein bind to CNX with sub-picomolar affinity, i.e., K D <1×10 -12 Binds to CNX at M.

[0187] In some embodiments, the antigen-binding molecules described herein have a K of 10 μM or less, preferably one of <5 μM, <2 μM, <1 μM, <500 nM, <100 nM, <75 nM, <50 nM, <40 nM, <30 nM, <20 nM, <15 nM, <12.5 nM, <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM, <3 nM, <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <50 pM, <40 pM, <30 pM, <20 pM, <10 pM, or <1 pM. D In some embodiments, the antigen-binding molecules described herein bind to human CNX at a K of 100 nM or less, preferably one of ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦15 nM, ≦12.5 nM, ≦10 nM, ≦9 nM, ≦8 nM, ≦7 nM, ≦6 nM, ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦500 pM, ≦400 pM, ≦300 pM, ≦200 pM, ≦100 pM, ≦50 pM, ≦40 pM, ≦30 pM, ≦20 pM, ≦10 pM, or ≦1 pM. D binds to human CNX at 100 ng / mL (as determined, for example, by the analysis described in Example 2 herein).

[0188] In some embodiments, the antigen-binding molecules described herein have an EC of 10 μM or less, preferably one of <5 μM, <2 μM, <1 μM, <500 nM, <100 nM, <75 nM, <50 nM, <40 nM, <30 nM, <20 nM, <15 nM, <12.5 nM, <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM, <3 nM, <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <50 pM, <40 pM, <30 pM, <20 pM, <10 pM, or <1 pM. 50binds to human CNX at 100 ng / mL (as determined, for example, by the analysis described in Example 2 herein).

[0189] In some embodiments, the antigen-binding molecule is cross-reactive with human CNX and its homologs (e.g., mouse CNX). In some embodiments, the antigen-binding molecule is cross-reactive with CNX and CRT. As used herein, a "cross-reactive" antigen-binding molecule / domain binds to a target antigen to which the antigen-binding molecule / domain is cross-reactive. For example, an antigen-binding molecule / domain / polypeptide that is cross-reactive with human CNX and mouse CNX can bind to both human CNX and mouse CNX. Similarly, an antigen-binding molecule / domain / polypeptide that is cross-reactive with human CNX and human CRT can bind to both CNX and CRT. A cross-reactive antigen-binding molecule / domain / polypeptide can exhibit specific binding to each of the target antigens.

[0190] In some embodiments, the antigen-binding molecule binds to human CNX (e.g., isoform 1) and mouse CNX. In some embodiments, the antigen-binding molecule binds to human CNX (e.g., isoform 1) and human CRT.

[0191] Antigen-binding molecules according to the present disclosure can bind to specific regions of interest on CNX. Antigen-binding molecules according to the present disclosure can bind to linear epitopes on CNX consisting of a continuous sequence of amino acids (i.e., a primary sequence of amino acids). In some embodiments, antigen-binding molecules can bind to conformational epitopes on CNX consisting of discontinuous sequences of amino acids in the amino acid sequence.

[0192] The region of a given target molecule to which an antigen-binding molecule binds can be determined by one of ordinary skill in the art using a variety of methods well known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometric hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire contents of which are incorporated herein by reference. In a preferred embodiment, the region of a peptide / polypeptide to which an antigen-binding molecule binds is determined by mass spectrometric hydrogen-deuterium exchange analysis performed essentially as described in Example 2 herein.

[0193] In some embodiments, the antigen-binding molecules of the present disclosure bind to a domain of CNX described herein, such as the luminal domain (e.g., lectin domain 1, P domain, lectin domain 2), the transmembrane domain, or the cytoplasmic domain.

[0194] In some embodiments, the antigen-binding molecule of the present disclosure binds to the luminal domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 337. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 337.

[0195] In some embodiments, the antigen-binding molecule of the present disclosure binds to the lectin domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 340. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 340.

[0196] In some embodiments, the antigen-binding molecule of the present disclosure binds to the P domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 341. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 341.

[0197] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 361. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 361.

[0198] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 362. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 362.

[0199] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 363. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 363.

[0200] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 364. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 364.

[0201] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 365. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 365.

[0202] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 366. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 366.

[0203] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 367. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 367.

[0204] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 368. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 368.

[0205] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 369. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 369.

[0206] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 370. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 370.

[0207] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 371. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 371.

[0208] In some embodiments, the antigen-binding molecule binds to the region of CNX set forth in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule contacts the region of CNX set forth in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 372. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 372.

[0209] In some embodiments, the antigen-binding molecule binds to a region of CNX set forth in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule contacts a region of CNX set forth in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule binds to CNX through contact with one or more amino acids in the region set forth in SEQ ID NO: 373. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 373.

[0210] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[0211] In some embodiments, the antigen-binding molecule can bind the same region of CNX or an overlapping region of CNX to a region of CNX bound by an antibody comprising the VL and VH regions of one of clones 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046, and C117 (see, e.g., Table C). In some embodiments, the antigen-binding molecule can bind the same region of CNX or an overlapping region of CNX to a region of CNX bound by an antibody comprising the VH and VL regions of C008 or 1E1.

[0212] Whether a test antigen-binding molecule binds to the same or overlapping region of a given target as a reference antigen-binding molecule can be evaluated, for example, by analyzing (i) the interaction between the test antigen-binding molecule and the target in the absence of the reference antigen-binding molecule, and (ii) the interaction between the test antigen-binding molecule and the target in the presence of the reference antigen-binding molecule or after incubating the target with the reference antigen-binding molecule. After analysis by (ii) compared to (i), determining a reduction in the level of interaction between the test antigen-binding molecule and the target can support the inference that the test antigen-binding molecule and the reference antigen-binding molecule bind to the same or overlapping region of the target. Suitable assays for such analysis include, for example, competitive ELISA assays and epitope binning assays.

[0213] In some embodiments, the antigen-binding molecule is an antagonist of CNX, CRT, and / or an antagonist of a complex comprising CNX or CRT. In some embodiments, the antigen-binding molecule can inhibit a function or process mediated by CNX and / or CRT, or a complex comprising CNX / CRT. In some embodiments, the antigen-binding molecule can inhibit a function or process mediated by a polypeptide complex comprising CNX or CRT. Here, "inhibition" refers to a decrease, reduction, or decrease relative to a control state. Suitable assays for examining the function of CNX and / or CRT and a complex comprising CNX / CRT are well known to those skilled in the art.

[0214] In some embodiments, the CNX-containing complex may be selected from a CNX:ERp57 complex, a CNX:ERp29 complex, and a CNX:CypB complex. In some embodiments, the CNX-containing complex may comprise CNX and a glycopolypeptide. In some embodiments, the CRT-containing complex may be selected from a CRT:ERp57 complex, a CRT:ERp29 complex, and a CRT:CypB complex. In some embodiments, the CRT-containing complex may comprise CRT and a glycopolypeptide.

[0215] In a preferred embodiment, the CNX-containing complex is a CNX:ERp57 complex. In a preferred embodiment, the CRT-containing complex is a CRT:ERp57 complex. Assays for identifying antigen-binding molecules capable of reducing / inhibiting the function of CNX / CRT and / or CNX / CRT-containing complexes may involve treating cells / tissues expressing CNX / CRT and / or CNX / CRT-containing complexes with the antigen-binding molecule to be tested, and then comparing the level of the relevant function to that observed in appropriate control conditions (e.g., untreated / vehicle-treated / control-treated cells / tissues).

[0216] Antigen-binding molecules capable of reducing / inhibiting the function of CNX / CRT and / or CNX / CRT-comprising complexes may be identified using an assay comprising detecting the level of a factor that correlates with the function of CNX / CRT and / or CNX / CRT-comprising complexes (e.g., gene and / or protein expression and / or activity of one or more proteins whose expression is directly / indirectly up- or down-regulated as a consequence of the function of CNX / CRT and / or CNX / CRT-comprising complexes). Such an assay may comprise treating cells / tissues expressing CNX / CRT and / or CNX / CRT-comprising complexes with the antigen-binding molecule, and subsequently (e.g., after an appropriate period of time, i.e., a period of time sufficient to observe the functional consequences of the activity of CNX / CRT and / or CNX / CRT-comprising complexes) comparing the level of the factor that correlates with the function of CNX / CRT and / or CNX / CRT-comprising complexes in such cells / tissues with the level of the factor that correlates with the relevant function in appropriate control conditions (e.g., untreated / vehicle-treated / control-treated cells / tissues).

[0217] In some embodiments, an antigen-binding molecule of the disclosure can reduce / inhibit a function of CNX / CRT or a complex comprising CNX / CRT by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of the relevant function observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule) in a given assay.

[0218] In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require or involve Fc-mediated function. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT independently of Fc-mediated function. That is, in some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT in an Fc region-independent manner.

[0219] The ability of an antigen-binding molecule to inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT through a mechanism that does not require or involve Fc-mediated function can be assessed, for example, by analyzing the ability of an antigen-binding molecule provided in a format lacking a functional Fc region to inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT. For example, the effect on the function of a complex comprising CNX / CRT and / or CNX / CRT can be examined using an antigen-binding molecule comprising a "silent" Fc region (e.g., containing a LALA PG substitution) or using an antigen-binding molecule provided in a format lacking an Fc region (e.g., scFv, Fab, etc.).

[0220] In some embodiments, the antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not involve ADCC. In some embodiments, the antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not involve ADCC. In some embodiments, the antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not involve CDC.

[0221] In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to an Fc receptor. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to an Fcγ receptor. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to FcγRIIIa. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to FcγRIIa. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to FcγRIIb. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to a complement protein. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to C1q. In some embodiments, an antigen-binding molecule can inhibit the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require N297 glycosylation.

[0222] It will be appreciated that in some embodiments, the antigen-binding molecules of the present disclosure achieve their functional effect through a mechanism that does not involve Fc-mediated function. In some embodiments, the antigen-binding molecules of the present disclosure achieve their functional effect through a mechanism that does not involve killing / depletion of cells expressing CNX / CRT or cells expressing complexes comprising CNX / CRT, e.g., Fc-mediated killing / depletion of such cells.

[0223] In some embodiments, the function of CNX / CRT or a complex comprising CNX / CRT may be selected from extracellular matrix (ECM) degradation, collagen degradation, gelatin degradation, oxidoreductase activity, and disulfide bond reductase activity. A factor correlated with the function of CNX / CRT or a complex comprising CNX / CRT may be, for example, ECM / collagen / gelatin degradation or production of oxidoreductase / disulfide bond reductase activity.

[0224] In some embodiments, the antigen-binding molecule reduces / inhibits extracellular matrix (ECM) degradation. In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation. In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation. In some embodiments, the antigen-binding molecule reduces / inhibits oxidoreductase activity. In some embodiments, the antigen-binding molecule reduces / inhibits disulfide bond reductase activity. In some embodiments, the antigen-binding molecule reduces / inhibits ECM degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits oxidoreductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits disulfide bond reductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex).

[0225] The ability of an antigen-binding molecule to inhibit ECM / collagen / gelatin degradation can be determined, for example, by analyzing ECM / collagen / gelatin degradation in the presence of or after incubation with the antigen-binding molecule. Antigen-binding molecules that can inhibit ECM / collagen / gelatin degradation are identified by observing a reduction / diminished level of ECM / collagen / gelatin degradation in the presence of the antigen-binding molecule (or after incubation with the antigen-binding molecule) compared to the level of ECM / collagen / gelatin degradation in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[0226] Antigen-binding molecules capable of reducing / inhibiting ECM / collagen / gelatin degradation (e.g., by CNX / CRT and / or complexes containing CNX / CRT) can be identified using an assay that includes detecting the level of ECM / collagen / gelatin or the level of a factor that correlates with ECM / collagen / gelatin degradation (e.g., products of degraded ECM / collagen / gelatin), e.g., using antibody-reporter-based methods. Assays for collagen / gelatin degradation are described, for example, in Hollander, Methods Mol. Biol. (2010) 622:367-78 and Vandooren et al., World J. Biol. Chem. (2011) 2(1):14-24. In a preferred embodiment, ECM / collagen / gelatin degradation may be assessed in an assay performed essentially as described in Example 4 herein.

[0227] For example, a commercially available gelatin solution (2%) can be labeled with 5-carboxy-X-rhodamine succinimidyl ester. The labeled gelatin can then be transferred to a sterile coverslip to create a thin layer and stabilized by glutaraldehyde fixation. A solution of rat tail collagen can be used to coat the coverslip, creating a thin layer of collagen on top of the gelatin. The coverslip can then be transferred to a culture vessel, and cells with appropriate degradation activity (e.g., human hepatocellular carcinoma Huh7 cells) can be seeded on the coverslip in the presence of the antigen-binding molecule to be tested and incubated for 48 hours to allow degradation to occur. The coverslip can then be fixed and subsequently stained with Hoechst to allow cell counting and analyzed by confocal microscopy. The acquired images can be analyzed using ImageJ to determine the surface of degraded gelatin and the total area per field. The number of nuclei can be calculated in parallel, and the final results can be normalized to the number of cells in each field.

[0228] For example, a mixture of rat tail collagen and quenched fluorescent DQ collagen type I can be coated and polymerized onto the bottom of a 384-well optical-grade plate. Cells of the 3t3-vSrc mouse cell line can be seeded on top of the collagen layer in the presence of the antigen-binding molecule to be tested and incubated for 48-72 hours. The fluorescent area of the DQ signal from live cells can then be assessed by high-content imaging and normalized by nuclei counting to determine the resolved area / cell.

[0229] In some embodiments, the antigen-binding molecules of the disclosure can reduce / inhibit ECM degradation, collagen degradation, or gelatin degradation by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of ECM degradation / collagen degradation / gelatin degradation observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule) in a given assay.

[0230] The ability of an antigen-binding molecule to inhibit the activity of an oxidoreductase can be determined, for example, by analyzing the activity of the oxidoreductase in the presence of, or after incubation with, the antigen-binding molecule. Antigen-binding molecules capable of inhibiting the activity of an oxidoreductase are identified by observing a reduction / decreased level of oxidoreductase activity in the presence of the antigen-binding molecule (or after incubation with the antigen-binding molecule) compared to the level of oxidoreductase activity in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[0231] Oxidoreductase activity can be assessed using any one of several methods known to those of skill in the art. For example, oxidoreductase activity can be assessed using the insulin reduction assay described, for example, in Hirano et al., Eur J Biochem. (1995) 234(1):336-42.

[0232] In some embodiments, an antigen-binding molecule of the disclosure can reduce / inhibit oxidoreductase activity by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of oxidoreductase activity observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in a given assay.

[0233] The ability of an antigen-binding molecule to inhibit the activity of disulfide bond reductase can be determined, for example, by analyzing the activity of disulfide bond reductase in the presence of, or after incubation with, the antigen-binding molecule. Antigen-binding molecules that can inhibit the activity of disulfide bond reductase are identified by observing a reduction / decrease in the level of disulfide bond reductase activity in the presence of, or after incubation with, the antigen-binding molecule, compared to the level of disulfide bond reductase activity in the absence of, or in the presence of, an appropriate control antigen-binding molecule.

[0234] The activity of disulfide bond reductase can be assessed using any one of several methods known to those skilled in the art. For example, disulfide bond reductase assays can employ antibodies for detecting the reduction of disulfide bonds in proteins, such as antibody clone OX133, which recognizes N-ethylmaleimide (NEM)-modified cysteine residues commonly present in polypeptides (see Holbrook et al., Mabs (2016) 8(4):672-677).

[0235] In some aspects, an antigen-binding molecule of the disclosure can reduce / inhibit disulfide bond reductase activity by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of disulfide bond reductase activity observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in a given assay.

[0236] In some embodiments, antigen-binding molecules according to the present disclosure reduce / inhibit cartilage degradation. Antigen-binding molecules capable of reducing / inhibiting cartilage degradation (e.g., by CNX / CRT and / or complexes containing CNX / CRT) may be identified using an assay that includes detecting cartilage levels or levels of factors correlated with cartilage degradation (e.g., products of degraded cartilage), e.g., using antibody-reporter-based methods. Cartilage degradation can be assessed essentially as described in Example 6 herein. Ex vivo assays for cartilage degradation are also described, for example, in Neidlin et al., PLoS One (2019) 14(10):e0224231.

[0237] In some aspects, antigen-binding molecules of the disclosure are capable of reducing / inhibiting cartilage degradation by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of cartilage degradation observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in a given assay.

[0238] In some aspects, antigen-binding molecules according to the present disclosure may enhance (i.e., upregulate, enhance) cell killing of cells containing / expressing CNX / CRT or a CNX / CRT-comprising complex. In some aspects, antigen-binding molecules according to the present disclosure may inhibit the growth of or reduce metastasis of cancers comprising cells containing / expressing CNX / CRT or a CNX / CRT-comprising complex. In some aspects, antigen-binding molecules according to the present disclosure may enhance (i.e., upregulate, enhance) cell killing of cells containing / expressing CNX / CRT or a CNX / CRT-comprising complex. In some aspects, antigen-binding molecules according to the present disclosure may inhibit the growth of or reduce metastasis of cancers comprising cells containing / expressing CNX / CRT or a CNX / CRT-comprising complex.

[0239] Cell killing can be assessed using, for example, any of the methods outlined in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro assays of cytotoxicity / cell killing include release assays, e.g. 51These assays include the Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed, for example, by co-culturing the test cells with the effector immune cells and measuring the number / proportion of live / dead (e.g., lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolysis in vitro capacity assay described in Cerignoli et al., PLoS One. (2018) 13(3):e0193498 (incorporated herein by reference in its entirety). Increased resistance to cell killing by granzyme B-expressing cells (e.g., effector immune cells) and / or decreased susceptibility to cell killing by such cells can be determined by detecting a decrease in the number / percentage of dead (e.g., lysed) test cells and / or an increase in the number / percentage of surviving (e.g., viable, non-lysed) test cells relative to a reference level of cell killing (e.g., for that cell type) after a given time.

[0240] In some embodiments, antigen-binding molecules according to the present disclosure can reduce the number / proportion of cells expressing CNX / CRT or a complex comprising CNX / CRT. In some embodiments, antigen-binding molecules according to the present disclosure can reduce the number / proportion of cells expressing CNX / CRT or a complex comprising CNX / CRT. In some embodiments, antigen-binding molecules according to the present disclosure can deplete or enhance the depletion of such cells.

[0241] The antigen-binding molecules of the present disclosure may contain one or more moieties to enhance reduction in the number / proportion of cells expressing CNX / CRT or a complex containing CNX / CRT. For example, the antigen-binding molecules of the present disclosure may contain, for example, an Fc region and / or a drug moiety.

[0242] The Fc region provides for interaction with Fc receptors and other molecules of the immune system, resulting in functional effects. IgG Fc-mediated effector functions are reviewed, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein by reference in its entirety), and include Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interaction of the Fc region with Fc receptors expressed by these cells, recruitment of complement pathway components through binding of the Fc region to the complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0243] In some embodiments, antigen-binding molecules according to the present disclosure comprise an Fc region that can enhance / direct one or more of ADCC, ADCP, CDC against cells expressing CNX / CRT or a complex comprising CNX / CRT (e.g., cells expressing CNX / CRT or a complex comprising CNX / CRT on their cell surface), and / or enhance MAC formation or cellular degranulation in these cells.

[0244] In some aspects, antigen-binding molecules according to the present disclosure can enhance / direct ADCC against cells expressing CNX / CRT or a complex comprising CNX / CRT. The ability and extent to which a given antigen-binding molecule can induce ADCC of a given target cell type can be determined by, for example, the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (incorporated herein by reference in its entirety), or by, for example, the method described in Jedema et al., Blood (2004) 103:2677-82 (incorporated herein by reference in its entirety). 51 The ability and extent of a given antigen-binding molecule to induce ADCP can be analyzed by a Cr release assay. The ability and extent of a given antigen-binding molecule to induce CDC can be analyzed, for example, by the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) pp. 157.17 (incorporated herein by reference in its entirety). The ability and extent of a given antigen-binding molecule to induce CDC can be analyzed by a C1q binding assay, for example, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29 (10): pp. 457-466 (incorporated herein by reference in its entirety).

[0245] In some embodiments, the antigen-binding molecules of the present disclosure do not induce ADCC of cells expressing CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecules do not induce ADCP of cells expressing CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecules do not induce CDC of cells expressing CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecules do not induce ADCC, ADCP, or CDC of cells expressing CNX / CRT or a complex comprising CNX / CRT on the cell surface.

[0246] An antigen-binding molecule that does not induce (i.e., is unable to induce) ADCC / ADCP / CDC does not induce substantially ADCC / ADCP / CDC activity against a relevant cell type, as determined, for example, by analysis in a suitable assay for the relevant activity. "Substantially no ADCC / ADCP / CDC activity" refers to a level of ADCC / ADCP / CDC that is not significantly greater than the ADCC / ADCP / CDC determined for an appropriate negative control molecule in a given assay (e.g., an antigen-binding molecule lacking an Fc region or an antigen-binding molecule comprising a "silent" Fc region (e.g., as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, incorporated by reference above). "Substantially no activity" can be a level of relevant activity that is ≦5-fold, e.g., ≦4-fold, ≦3-fold, ≦2.5-fold, ≦2-fold, or ≦1.5-fold the level of activity determined for an appropriate negative control molecule in a given assay.

[0247] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a drug moiety. The antigen-binding molecule may be conjugated to a drug moiety. Antibody-drug conjugates are generally described, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety is or comprises a cytotoxic agent, such that the antigen-binding molecule exhibits cytotoxicity against cells expressing CNX / CRT or a complex comprising CNX / CRT (e.g., cells expressing CNX / CRT or a complex comprising CNX / CRT on the cell surface). In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0248] In some embodiments, an antigen-binding molecule according to the present disclosure comprises an immune cell-engaging portion. In some embodiments, an antigen-binding molecule comprises a CD3 polypeptide-binding portion (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[0249] In some aspects, antigen-binding molecules according to the present disclosure can enhance / direct T-cell mediated cytotoxic activity against cells expressing CNX / CRT or a complex comprising CNX / CRT.

[0250] In some embodiments, the antigen-binding molecules of the present disclosure exhibit anti-cancer activity. In some embodiments, the antigen-binding molecules of the present disclosure increase the killing of cancer cells. In some embodiments, the antigen-binding molecules of the present disclosure cause a reduction in the number of cancer cells in vivo, for example, compared to an appropriate control condition. The cancer may be a cancer that expresses CNX / CRT or a complex containing CNX / CRT.

[0251] In some embodiments, antigen-binding molecules according to the present disclosure reduce / inhibit cancer and / or cancer tumor growth. In some embodiments, antigen-binding molecules reduce tissue invasion by cancer cells. In some embodiments, antigen-binding molecules reduce cancer metastasis. In some embodiments, antigen-binding molecules exhibit anti-cancer activity. In some embodiments, antigen-binding molecules reduce cancer cell growth / proliferation. In some embodiments, antigen-binding molecules reduce cancer cell survival. In some embodiments, antigen-binding molecules increase cancer cell killing. In some embodiments, antigen-binding molecules of the present disclosure cause a reduction in the number of cancer cells, for example in vivo. The cancer may be a cancer comprising cells expressing CNX and / or CRT.

[0252] Antigen-binding molecules of the present disclosure may be analyzed for the properties described in the preceding paragraphs in suitable assays, including, for example, in vivo models, such as those performed essentially as described in Example 5 herein.

[0253] In some embodiments, administration of an antigen-binding molecule according to the present disclosure may result in one or more of: inhibiting cancer development / progression, delaying / preventing cancer onset, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, reducing the severity of cancer symptoms, reducing the number of cancer cells, reducing the size / volume of the tumor, and / or increasing survival (e.g., progression-free survival or overall survival) as determined, for example, in a suitable model.

[0254] In some embodiments, an antigen-binding molecule of the disclosure can reduce / inhibit tumor growth (e.g., in an in vivo model of, e.g., liver cancer) by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the tumor growth observed in the absence of treatment with the antigen-binding molecule (or following treatment with a suitable control antigen-binding molecule known to have no effect on tumor growth) in a given assay.

[0255] In some embodiments, an antigen-binding molecule of the disclosure can reduce / inhibit metastasis (e.g., in an in vivo model of breast cancer metastasis to the lung) by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of metastasis observed in the absence of treatment with the antigen-binding molecule in a given assay (or following treatment with a suitable control antigen-binding molecule known to have no effect on metastasis).

[0256] In some embodiments, an antigen-binding molecule of the disclosure can increase survival of a subject with cancer (e.g., in an in vivo model of, e.g., liver cancer or breast cancer) by more than 1-fold, e.g., one of the following: ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, the level of survival observed in a given assay in the absence of treatment with the antigen-binding molecule (or following treatment with a suitable control antigen-binding molecule known to have no effect on survival).

[0257] In some embodiments, the antigen binding molecules according to the present disclosure reduce / inhibit the pathology of a disease / condition characterized by ECM degradation in a subject. In some embodiments, antigen-binding molecules according to the present disclosure reduce / inhibit the pathology of a disease / condition characterized by cartilage degradation (e.g., arthritis) in a subject. In some embodiments, antigen-binding molecules according to the present disclosure reduce the arthritis score in a subject with arthritis. Arthritic pathology may be assessed in an assay performed in a suitable in vivo model known to those of skill in the art. Such models include, for example, the murine collagen antibody-induced arthritis (CAIA) model described in Khachigian, Nat Protoc. (2006) 1(5):2512-6; such assays may be performed essentially as described in Example 5 or Example 6 herein. In some embodiments, subjects treated with antigen-binding molecules according to the present disclosure are determined to have a lower arthritis score (e.g., on day 7, 8, 9, or 10) compared to subjects not treated with the antigen-binding molecule (or compared to subjects treated with a suitable control antigen-binding molecule known not to affect arthritis pathology).

[0258] In some embodiments, the antigen-binding molecules of the disclosure are capable of reducing / inhibiting the pathology of a disease / condition (e.g., arthritis) characterized by ECM degradation or cartilage degradation in a subject in a given assay (e.g., as determined by arthritis score, e.g., in a CAIA model) by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level observed in the absence of treatment with the antigen-binding molecule (or following treatment with a suitable control antigen-binding molecule known to have no effect on the pathology of the disease / condition).

[0259] The antigen-binding molecules of the present disclosure preferably possess novel and / or improved properties compared to known antigen-binding molecules that bind to CNX. Known antibodies against CNX include monoclonal antibodies clone AF18 (Invitrogen Cat. No. MA3-027), clone AF8 (Merck Cat. No. MABF2067), clone TO-5 (Merck Cat. No. C7617), clone 3H4A7 (Invitrogen Cat. No. MA5-15389), clone ARC0648 (Invitrogen Cat. No. MA5-35588), clone GT1563 (GeneTex Cat. No. GTX629976), clone CANX / 1541 (GeneTex Cat. No. GTX34446), clone IE2.1C12 (Novus Biologicals Cat. No. NBP2-36571), clone 1C2.2D11 (Novus Biologicals Cat. No. NBP2-36570SS), and clone 2A2C6 (Proteintech Cat. No. 66903-1-Ig), clone C5C9 (Cell Signaling Technology, Inc. Cat. No. 2679), clone E-10 (Santa Cruz Biotechnology Cat. No. sc-46669), polyclonal antibodies ab10286 and ab22595 (Abcam), and the anti-CNX antibody disclosed in CN 101659702 A (e.g., the antibody produced by hybridoma CGMCC No. 3240). In some embodiments, the known antibody against CNX is polyclonal antibody ab10286.

[0260] In some aspects, an antigen-binding molecule according to the present disclosure comprises: It binds to CNX (e.g., human CNX and / or mouse CNX) with greater affinity than known antibodies against CNX.

[0261] It binds to CRT (eg, human CRT) with greater affinity than known antibodies against CNX. It reduces the function of CNX / CRT or the function of a complex containing CNX / CRT to a greater extent than known antibodies against CNX.

[0262] It reduces extracellular matrix degradation (eg, collagen and / or gelatin degradation) to a greater extent / with a greater ability than known antibodies against CNX. It reduces oxidoreductase activity to a greater extent than known antibodies against CNX.

[0263] It reduces the activity of disulfide bond reductase to a greater extent than known antibodies against CNX. It reduces cartilage degradation to a greater extent than known antibodies against CNX.

[0264] It increases killing of cells expressing CNX / CRT to a greater extent than known antibodies to CNX. It increases ADCC of cells expressing CNX / CRT to a greater extent than known antibodies against CNX.

[0265] Inhibits tumor growth to a greater extent than known antibodies against CNX. Reduces cancer metastasis to a greater extent than known antibodies against CNX. Increases survival of subjects with cancer to a greater extent than known antibodies to CNX; and / or It reduces the pathology of diseases / conditions characterized by ECM degradation in a subject to a greater extent than known antibodies to CNX.

[0266] It reduces the pathology of diseases / conditions characterized by cartilage degradation (eg, arthritis) in a subject to a greater extent than known antibodies to CNX. In some embodiments, an antigen-binding molecule according to the present disclosure has an EC2 activity greater than that of known antibodies against CNX, as determined by a given assay. 50EC of less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 and binds to CNX (e.g., human CNX and / or mouse CNX).

[0267] In some embodiments, an antigen-binding molecule according to the present disclosure has a K value lower than that of known antibodies against CNX, as determined by a given assay. D or less than 1 time, for example, ≦0.99 times, ≦0.95 times, ≦0.9 times, ≦0.85 times, ≦0.8 times, ≦0.75 times, ≦0.7 times, ≦0.65 times, ≦0.6 times, ≦0.55 times, ≦0.5 times, ≦0.45 times, ≦0.4 times, ≦0.35 times, ≦0.3 times, ≦0.25 times, ≦0.2 times, ≦0.15 times, ≦0.1 times, ≦0.05 times, or ≦0.01 times. D and binds to CNX (e.g., human CNX and / or mouse CNX).

[0268] In some embodiments, an antigen-binding molecule according to the present disclosure has an EC2 activity greater than that of known antibodies against CNX, as determined by a given assay. 50 EC of less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 and binds to CRT (e.g., human CRT).

[0269] In some embodiments, an antigen-binding molecule according to the present disclosure has a K value lower than that of known antibodies against CNX, as determined by a given assay. Dor less than 1 time, for example, ≦0.99 times, ≦0.95 times, ≦0.9 times, ≦0.85 times, ≦0.8 times, ≦0.75 times, ≦0.7 times, ≦0.65 times, ≦0.6 times, ≦0.55 times, ≦0.5 times, ≦0.45 times, ≦0.4 times, ≦0.35 times, ≦0.3 times, ≦0.25 times, ≦0.2 times, ≦0.15 times, ≦0.1 times, ≦0.05 times, or ≦0.01 times. D and binds to CRT (e.g., human CRT).

[0270] In some embodiments, an antigen-binding molecule according to the present disclosure has an IC value higher than that of a known antibody against CNX, as determined by a given assay. 50 less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 and reduces the function of CNX / CRT and / or the function of a complex containing CNX / CRT.

[0271] In some embodiments, an antigen-binding molecule according to the present disclosure reduces the function of CNX / CRT and / or the function of a complex comprising CNX / CRT in a given assay by less than 1-fold the level at which the function is reduced by a known antibody to CNX at a comparable concentration, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0272] In some embodiments, an antigen-binding molecule according to the present disclosure has an IC value higher than that of a known antibody against CNX, as determined by a given assay. 50less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 and reducing extracellular matrix degradation, collagen degradation, and / or gelatin degradation.

[0273] In some embodiments, an antigen-binding molecule according to the present disclosure reduces extracellular matrix degradation, collagen degradation, and / or gelatin degradation in a given assay by less than 1-fold the level at which ECM / collagen / gelatin degradation is reduced by a known antibody to CNX at a comparable concentration, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0274] In some embodiments, an antigen-binding molecule according to the present disclosure has an IC value higher than that of a known antibody against CNX, as determined by a given assay. 50 less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 This reduces the activity of oxidoreductase.

[0275] In some embodiments, an antigen-binding molecule according to the present disclosure reduces oxidoreductase activity in a given assay by less than 1-fold the level at which a known antibody to CNX at a comparable concentration reduces oxidoreductase activity, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0276] In some embodiments, an antigen-binding molecule according to the present disclosure has an IC value higher than that of a known antibody against CNX, as determined by a given assay. 50 less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 and reduces the activity of disulfide bond reductase.

[0277] In some embodiments, an antigen-binding molecule according to the present disclosure reduces disulfide bond reductase activity in a given assay by less than 1-fold the level at which a known antibody to CNX at a comparable concentration reduces disulfide bond reductase activity, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0278] In some embodiments, an antigen-binding molecule according to the present disclosure has an IC value higher than that of a known antibody against CNX, as determined by a given assay. 50less than 1x, for example, ≦0.99x, ≦0.95x, ≦0.9x, ≦0.85x, ≦0.8x, ≦0.75x, ≦0.7x, ≦0.65x, ≦0.6x, ≦0.55x, ≦0.5x, ≦0.45x, ≦0.4x, ≦0.35x, ≦0.3x, ≦0.25x, ≦0.2x, ≦0.15x, ≦0.1x, ≦0.05x, or ≦0.01x 50 This reduces cartilage breakdown.

[0279] In some embodiments, an antigen-binding molecule according to the present disclosure reduces cartilage degradation in a given assay by less than 1-fold the level at which cartilage degradation is reduced by a known antibody to CNX at a comparable concentration, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0280] In some embodiments, an antigen-binding molecule according to the present disclosure increases killing or ADCC of cells expressing CNX / CRT and / or a complex comprising CNX / CRT by more than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, the level of killing / ADCC achieved by treatment with a comparable concentration of a known antibody to CNX in a given assay.

[0281] In some embodiments, an antigen-binding molecule according to the present disclosure reduces / inhibits tumor growth (e.g., in an in vivo model of liver cancer) in a given assay to less than 1-fold, e.g., one of the following levels: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold the level at which tumor growth is inhibited by treatment with a known antibody to CNX at a comparable concentration.

[0282] In some embodiments, an antigen-binding molecule according to the disclosure reduces metastasis (e.g., in an in vivo model of breast cancer metastasis to the lung) in a given assay to less than 1-fold the level at which metastasis is reduced by treatment with a known antibody to CNX at a comparable concentration, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold.

[0283] In some embodiments, an antigen-binding molecule according to the present disclosure increases the survival rate of a subject with cancer in a given assay by more than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, the level of survival achieved by treatment with a known antibody against CNX at a comparable concentration.

[0284] In some embodiments, an antigen-binding molecule according to the present disclosure reduces lesions of a disease / condition (e.g., arthritis) characterized by ECM degradation or cartilage degradation in a subject (e.g., as determined by arthritis score, e.g., in a CAIA model) in a given assay to less than 1-fold the level at which lesions are inhibited by treatment with a known antibody to CNX at a comparable concentration, e.g., by one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold. Chimeric antigen receptor (CAR) The present disclosure also provides an antigen-binding polypeptide or a chimeric antigen receptor (CAR) comprising a polypeptide of the present disclosure.

[0285] CAR is a recombinant receptor that provides both antigen binding and T cell activation functions.The structure and engineering of CAR are outlined in, for example, Dotti et al., Immunol Rev (2014) 257 (1), the entirety of which is incorporated herein by reference.CAR comprises an antigen binding region and a signal region linked to a cell membrane anchor region.An optional hinge region provides separation between the antigen binding region and the cell membrane anchor region and can act as a flexible linker.

[0286] A CAR of the present disclosure comprises an antigen-binding region that comprises or consists of an antigen-binding molecule of the present disclosure, or that comprises or consists of a polypeptide of the present disclosure. A cell membrane anchor region is provided between the antigen binding region and the signaling region of the CAR, anchoring the CAR to the cell membrane of a cell expressing the CAR, with the antigen binding region in the extracellular space and the signaling region intracellularly. In some embodiments, the CAR comprises a cell membrane anchor region that comprises or consists of, or comprises or consists of, the amino acid sequence of the transmembrane region of one of CD3-zeta, CD4, CD8, or CD28. As used herein, a region "derived" from a reference amino acid sequence comprises an amino acid sequence that has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0287] The signaling region of a CAR enables T cell activation. The signaling region of a CAR may comprise the amino acid sequence of the intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signaling regions containing sequences from other ITAM-containing proteins, such as FcγRI, have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1):182-187). The signaling region of a CAR may comprise a costimulatory sequence derived from the signaling region of a costimulatory molecule to facilitate activation of CAR-expressing T cells upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CARs are engineered to provide costimulation of various intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB-mediated signaling is via TNF receptor-associated factor (TRAF) adaptor proteins. Thus, the signaling region of a CAR may comprise costimulatory sequences derived from the signaling regions of two or more costimulatory molecules. In some embodiments, the CARs of the present disclosure comprise one or more costimulatory sequences that comprise or consist of, or comprise or consist of, the amino acid sequence of one or more intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27.

[0288] The optional hinge region provides separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. The hinge region is derived from IgG1. In some embodiments, the CAR of the present disclosure comprises a hinge region that comprises or consists of the amino acid sequence of the hinge region of IgG1, or that comprises or consists of the amino acid sequence derived therefrom.

[0289] Also provided are cells comprising CARs according to the present disclosure. CARs according to the present disclosure can be used to generate immune cells that express CARs, such as CAR-T cells or CAR-NK cells. CARs can be engineered into immune cells during in vitro culture.

[0290] The antigen-binding region of the CAR of the present disclosure may be provided in any suitable format, e.g., scFv, scFab, etc. Nucleic acids and vectors The present disclosure provides a nucleic acid or multiple nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0291] In some embodiments, the nucleic acid may be or be contained in a vector or vectors. That is, the nucleotide sequence of the nucleic acid may be contained in a vector. The antigen-binding molecule, polypeptide, or CAR according to the present disclosure is produced intracellularly by transcription from a vector encoding the antigen-binding molecule, polypeptide, or CAR, followed by translation of the transcribed RNA.

[0292] Therefore, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate the delivery of a nucleic acid encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. The vector may be an expression vector containing the elements necessary to express a nucleic acid comprising / encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure.

[0293] The nucleic acids and vectors according to the present disclosure are provided in purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials. The nucleotide sequence may be contained in a vector, for example, an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acids into cells. The vector may be a vector for expressing nucleic acids in cells. Such a vector may contain a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also contain a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0294] The term "operably linked" can include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked such that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). That is, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of effecting transcription of the nucleic acid sequence. The resulting transcript is then translated into the desired peptide / polypeptide.

[0295] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., retroviral vectors, e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), e.g., as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated by reference in their entirety.

[0296] In some embodiments, the vector can be a eukaryotic vector, e.g., a vector that contains elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector can be a mammalian vector that includes, e.g., a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0297] The constituent polypeptides of an antigen-binding molecule according to the present disclosure may be encoded by different nucleic acids from the plurality of nucleic acids or different vectors from the plurality of vectors. Antigen-binding molecules and cells containing / expressing polypeptides The present disclosure also provides a cell comprising or expressing an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure.

[0298] The cell may be a eukaryotic cell, e.g., a mammalian cell. The mammal may be a primate (such as a rhesus monkey, a cynomolgus monkey, a non-human primate, or a human), or a non-human mammal (e.g., a rabbit, a guinea pig, a rat, a mouse, or other rodent (including any animal in the order Rodentia), a cat, a dog, a pig, a sheep, a goat, a cow (including a cow, e.g., a dairy cow, or any animal in the order Bovidae), a horse (including any animal in the order Equidae), a donkey, and a non-human primate).

[0299] In some embodiments, the cells are or are derived from cell types commonly used for the expression of polypeptides for use in human therapy. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (incorporated herein by reference in its entirety), and include, for example, CHO, HEK 293, PER.C6, NS0, and BHK cells. In preferred embodiments, the cells are or are derived from CHO cells.

[0300] The present disclosure also provides a method of producing a cell comprising a nucleic acid or vector according to the present disclosure, the method comprising introducing into the cell a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure. In some aspects, introducing into the cell an isolated nucleic acid or vector according to the present disclosure comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0301] The present disclosure also provides a method for producing a cell that expresses / contains an antigen-binding molecule, polypeptide, or CAR according to the present disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure into the cell. In some aspects, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some aspects, the method is performed in vitro.

[0302] The present disclosure also provides cells obtained or obtainable by a method according to the present disclosure. Production of antigen-binding molecules and polypeptides Antigen-binding molecules and polypeptides according to the present disclosure may be prepared according to methods for the production of polypeptides well known to those skilled in the art.

[0303] Polypeptides may be prepared by chemical synthesis, e.g., liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in Chandrudu et al., Molecules (2013), 18:4373-4388, the entire contents...

Claims

1. An optionally isolated antigen-binding molecule that binds to CNX.

2. The antigen-binding molecule of claim 1, which inhibits the degradation of extracellular matrix (ECM).

3. (a) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 166; HC-CDR2 having the amino acid sequence of SEQ ID NO: 167; HC-CDR3 having the amino acid sequence of SEQ ID NO: 168 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 179, LC-CDR2 having the amino acid sequence of SEQ ID NO: 180, LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 a light chain variable (VL) region incorporating (b) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33; HC-CDR2 having the amino acid sequence of SEQ ID NO: 34; HC-CDR3 having the amino acid sequence of SEQ ID NO: 35 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41, LC-CDR2 having the amino acid sequence of SEQ ID NO: 42, LC-CDR3 having the amino acid sequence of SEQ ID NO: 43 a light chain variable (VL) region incorporating (c) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO:4 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 10, LC-CDR2 having the amino acid sequence of SEQ ID NO: 11, LC-CDR3 having the amino acid sequence of SEQ ID NO: 12 a light chain variable (VL) region incorporating (d) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 18; HC-CDR2 having the amino acid sequence of SEQ ID NO: 19; HC-CDR3 having the amino acid sequence of SEQ ID NO: 20 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 25, LC-CDR2 having the amino acid sequence of SEQ ID NO: 26, LC-CDR3 having the amino acid sequence of SEQ ID NO: 27 a light chain variable (VL) region incorporating (e) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO:49 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 53, LC-CDR2 having the amino acid sequence of SEQ ID NO: 54, LC-CDR3 having the amino acid sequence of SEQ ID NO: 55 a light chain variable (VL) region incorporating (f) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 68, LC-CDR2 having the amino acid sequence of SEQ ID NO: 26, LC-CDR3 having the amino acid sequence of SEQ ID NO: 69 a light chain variable (VL) region incorporating (g) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73, LC-CDR2 having the amino acid sequence of SEQ ID NO: 26, LC-CDR3 having the amino acid sequence of SEQ ID NO: 74 a light chain variable (VL) region incorporating (h) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 62; HC-CDR3 having the amino acid sequence of SEQ ID NO: 63 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 78, LC-CDR2 having the amino acid sequence of SEQ ID NO: 79, LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 a light chain variable (VL) region incorporating (i) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 83 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73, LC-CDR2 having the amino acid sequence of SEQ ID NO: 26, LC-CDR3 having the amino acid sequence of SEQ ID NO: 74 a light chain variable (VL) region incorporating (j) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 86 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 89, LC-CDR2 having the amino acid sequence of SEQ ID NO: 11, LC-CDR3 having the amino acid sequence of SEQ ID NO: 90 a light chain variable (VL) region incorporating (k) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 61; HC-CDR2 having the amino acid sequence of SEQ ID NO: 95; HC-CDR3 having the amino acid sequence of SEQ ID NO: 96 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 101, LC-CDR2 having the amino acid sequence of SEQ ID NO: 102, LC-CDR3 having the amino acid sequence of SEQ ID NO: 103 a light chain variable (VL) region incorporating (l) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108; HC-CDR2 having the amino acid sequence of SEQ ID NO: 109; HC-CDR3 having the amino acid sequence of SEQ ID NO: 110 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 115, LC-CDR2 having the amino acid sequence of SEQ ID NO: 116, LC-CDR3 having the amino acid sequence of SEQ ID NO: 117 a light chain variable (VL) region incorporating (m) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 122 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 125, LC-CDR2 having the amino acid sequence of SEQ ID NO: 126, LC-CDR3 having the amino acid sequence of SEQ ID NO: 127 a light chain variable (VL) region incorporating (n) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 132; HC-CDR2 having the amino acid sequence of SEQ ID NO: 133; HC-CDR3 having the amino acid sequence of SEQ ID NO: 134 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 139, LC-CDR2 having the amino acid sequence of SEQ ID NO: 140, LC-CDR3 having the amino acid sequence of SEQ ID NO: 80 a light chain variable (VL) region incorporating (o) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 146; HC-CDR2 having the amino acid sequence of SEQ ID NO: 147; HC-CDR3 having the amino acid sequence of SEQ ID NO: 148 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41, LC-CDR2 having the amino acid sequence of SEQ ID NO: 42, LC-CDR3 having the amino acid sequence of SEQ ID NO: 153 a light chain variable (VL) region incorporating (p) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 3; HC-CDR3 having the amino acid sequence of SEQ ID NO: 156 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 158, LC-CDR2 having the amino acid sequence of SEQ ID NO: 159, LC-CDR3 having the amino acid sequence of SEQ ID NO: 160 a light chain variable (VL) region incorporating (q) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 166; HC-CDR2 having the amino acid sequence of SEQ ID NO: 167; HC-CDR3 having the amino acid sequence of SEQ ID NO: 168 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 171, LC-CDR2 having the amino acid sequence of SEQ ID NO: 172, LC-CDR3 having the amino acid sequence of SEQ ID NO: 173 a light chain variable (VL) region incorporating (r) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 186; HC-CDR3 having the amino acid sequence of SEQ ID NO: 187 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 73, LC-CDR2 having the amino acid sequence of SEQ ID NO: 26, LC-CDR3 having the amino acid sequence of SEQ ID NO: 194 a light chain variable (VL) region incorporating (s) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 199; HC-CDR3 having the amino acid sequence of SEQ ID NO: 200 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 205, LC-CDR2 having the amino acid sequence of SEQ ID NO: 42, LC-CDR3 having the amino acid sequence of SEQ ID NO: 206 a light chain variable (VL) region incorporating (t) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 211; HC-CDR3 having the amino acid sequence of SEQ ID NO: 212 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 216, LC-CDR2 having the amino acid sequence of SEQ ID NO: 172, LC-CDR3 having the amino acid sequence of SEQ ID NO: 217 a light chain variable (VL) region incorporating (u) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 222; HC-CDR2 having the amino acid sequence of SEQ ID NO: 223; HC-CDR3 having the amino acid sequence of SEQ ID NO: 224 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229, LC-CDR2 having the amino acid sequence of SEQ ID NO: 172, LC-CDR3 having the amino acid sequence of SEQ ID NO: 230 a light chain variable (VL) region incorporating (v) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 48; HC-CDR2 having the amino acid sequence of SEQ ID NO: 199; HC-CDR3 having the amino acid sequence of SEQ ID NO: 200 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235, LC-CDR2 having the amino acid sequence of SEQ ID NO: 236, LC-CDR3 having the amino acid sequence of SEQ ID NO: 237 a light chain variable (VL) region incorporating (lol) (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 185; HC-CDR2 having the amino acid sequence of SEQ ID NO: 243; HC-CDR3 having the amino acid sequence of SEQ ID NO: 244 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 248, LC-CDR2 having the amino acid sequence of SEQ ID NO: 249, LC-CDR3 having the amino acid sequence of SEQ ID NO: 250 A light chain variable (VL) region incorporating The antigen-binding molecule of claim 1 or 2, comprising:

4. a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165, 32, 1, 17, 47, 60, 82, 85, 94, 107, 121, 131, 154, 155, 184, 198, 210, 221, or 242; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 178, 40, 9, 24, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 191, 204, 215, 228, 234, or 247; Including, Optionally, (i) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 178; or (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 32; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 40; or (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:9; or (iv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (v) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 47; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 52; or (vi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 67; or (vii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 72; or (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 77; or (ix) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of a SEQ ID NO:; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:; or (x) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 82; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 72; or (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 85; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 88; or (xii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 94; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 100; or (xiii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 107; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 114; or (xiv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 121; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 124; or (xv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 131; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 138; or (xvi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 145; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 152; or (xvii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 155; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 157; or (xviii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 165; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 170; or (xix) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 184; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 191; or (xx) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 204; or (xxi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 210; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 215; or (xxii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 221; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 228; or (xxiii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 234; or (xxiv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 242; and A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

247. The antigen-binding molecule of claim 1 , comprising:

5. 5. The antigen-binding molecule of claim 1, which (a) binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 363, and optionally binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 361 or 362, or (b) binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 371, and optionally binds to CNX through contact with one or more amino acid residues in a region of CNX corresponding to the region set forth in SEQ ID NO: 364, 365, 366, 367, 368, 369, 370, 372, or 373.

6. The antigen-binding molecule of claim 1 , which binds to CRT.

7. The antigen-binding molecule of claim 1 , which binds to human CNX and mouse CNX.

8. The antigen-binding molecule of any one of claims 1 to 8, which is a multispecific antigen-binding molecule and further comprises an antigen-binding domain that binds to an antigen other than CNX.

9. The antigen-binding molecule of claim 8, wherein the multispecific antigen-binding molecule is a bispecific T cell engager (BiTE).

10. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule of any one of claims 1 to 9.

11. 10. An antibody drug conjugate (ADC) comprising the antigen-binding molecule of any one of claims 1 to 9 and a drug moiety.

12. 11. A nucleic acid or nucleic acids encoding the antigen-binding molecule of any one of claims 1 to 9 or the CAR of claim 10, optionally isolated.

13. 13. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 12.

14. A cell comprising the antigen-binding molecule of any one of claims 1 to 9, the CAR of claim 10, the ADC of claim 11, the nucleic acid or multiple nucleic acids of claim 12, or the expression vector or multiple expression vectors of claim 13.

15. 15. A method comprising culturing the cell of claim 14 under conditions suitable for expression of the antigen-binding molecule or CAR by the cell.

16. A composition comprising the antigen-binding molecule of any one of claims 1 to 9, the CAR of claim 10, the ADC of claim 11, the nucleic acid or nucleic acids of claim 12, the expression vector or expression vectors of claim 13, or the cell of claim 14, together with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

17. 17. An antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, an ADC according to claim 11, a nucleic acid or nucleic acids according to claim 12, an expression vector or expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16, for use in a method of medical treatment and prophylaxis.

18. 17. The antigen-binding molecule of any one of claims 1 to 9, the CAR of claim 10, the ADC of claim 11, the nucleic acid or nucleic acids of claim 12, the expression vector or expression vectors of claim 13, the cell of claim 14, or the composition of claim 16, for use in a method for treating or preventing a disease / condition characterized by extracellular matrix (ECM) degradation.

19. 17. The antigen-binding molecule of any one of claims 1 to 9, the CAR of claim 10, the ADC of claim 11, the nucleic acid or nucleic acids of claim 12, the expression vector or expression vectors of claim 13, the cell of claim 14, or the composition of claim 16, for use in a method for treating or preventing cancer.

20. 20. The antigen-binding molecule, CAR, ADC, nucleic acid or nucleic acids, expression vector or expression vectors, cell, or composition for use according to claim 19, wherein the cancer is selected from liver cancer, breast cancer, oral cancer, oral squamous cell carcinoma, sarcoma, lung cancer, prostate cancer, bladder cancer, renal cancer, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer, ovarian cancer, cervical cancer, brain cancer, bile duct cancer, testicular cancer, and thyroid cancer.

21. 17. The antigen-binding molecule of any one of claims 1 to 9, the CAR of claim 10, the ADC of claim 11, the nucleic acid or nucleic acids of claim 12, the expression vector or expression vectors of claim 13, the cell of claim 14, or the composition of claim 16, for use in a method for treating or preventing cartilage degradation or a disease / condition characterized by cartilage degradation.

22. 22. The antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell, or composition for use according to claim 21, wherein the disease / condition characterized by cartilage degradation is selected from joint disorders, arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondrosis dissecans, cartilage damage, and polychondritis.

23. 10. Use of an antigen-binding molecule according to any one of claims 1 to 9 to deplete or enhance killing of cells expressing CNX.

24. 10. An in vitro complex comprising an antigen-binding molecule of any one of claims 1 to 9, which binds to CNX, optionally isolated.

25. 10. A method for detecting CNX in a sample, comprising the steps of contacting a sample containing or suspected of containing CNX with an antigen-binding molecule according to any one of claims 1 to 9, and detecting the formation of a complex between the antigen-binding molecule and CNX.

26. 10. A method of selecting or stratifying a subject for treatment with an agent that targets CNX, the method comprising the steps of contacting a sample from the subject in vitro with an antigen-binding molecule of any one of claims 1 to 9, and detecting the formation of a complex between the antigen-binding molecule and CNX.

27. 10. Use of the antigen-binding molecule of any one of claims 1 to 9 as an in vitro or in vivo diagnostic or prognostic agent.

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